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Linz jor professor seza % gfimzfl- MS U is an Affirmative Action/Equal Opportunity Institution V V V V V PLACE Ii RETURN Boanmwombcinckomnom yourncord. TO AVOiD FINES Mum on or More duo duo. MSU In An Affinnltivo Action/Equal Opportunity IMW W1 \ (“It .i “HIKE!” " lTAJ‘I/x LIDHHWJIIO 3 7' 2/. - _‘.————.. ‘——.-—- a“. IDENTIFICATION AND ANALYSIS OF THE EXPRESSION OF THE AFLATOXIN BIOSYNTHETIC GENES NOR-1 and VER-l IN THE COMMERCIAL SPECIES ASPERGILL US SOJAE AND A. UK YZAE AS WELL As TOXIGENIC AND NON-TOXIGENIC STRAINS OF A. FLA VUS By Matthew David Rarick A Thesis Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Master of Science Department of Food Science and Human Nutrition 1996 Ci prod have aflab thea (con prod Pathx andi Confi then induc aflau Aflat Con“ Ihe A ABSTRACT IDENTIFICATION AND ANALYSIS OF THE EXPRESSION OF THE AFLATOXIN BIOSYNTHETIC GENES NOR-1 and VER-l IN THE COMMERCIAL SPECIES ASPERGILL US SOJAE AND A. 0R YZAE As WELL AS TOXIGENIC AND NON-TOXIGENIC STRAINS OF A. FLA VUS By Matthew David Rarick Aflatoxins are a group of highly potent, carcinogenic, secondary metabolites produced by the imperfect fiingi Aspergillusflavus and A. parasiticus. Previous studies have revealed that many species of Aspergillus have at least some of the genes involved in aflatoxin production and, in some cases, are able to use them to produce intermediates of the aflatoxin biosynthetic pathway. This study examined A. sojae (AS) and A. oryzae (A0) (commercial species used in enzyme production) as well as four A. flavus strains (aflatoxin producers 1059, 1273; non-producers 2112, 2115) for the presence of the aflatoxin pathway genes nor-1 and ver-l, for aflatoxin production, and for accumulation of nor-1 and ver-l transcripts. Southern hybridization with the DNA probes nor-1 and ver-l confirmed the presence of similar or identical genes in each strain. The fungal strains were then grown in duplicate to 48 and 72 hours as stationary and shake cultures in an aflatoxin inducing grth medium. The media from one set of duplicate cultures was analyzed for aflatoxin accumulation by means of enzyme linked immunosorbent assay (ELISA). Aflatoxin production by the A. flavus producers was detected, though at reduced levels compared to A. parasiticus SU—l, whereas, no toxin was ever detected from AS, A0, and the A flaws non-producers. The levels of aflatoxin produced in the stationary cultures . "\'1/ ~, fur F' -‘) )i i)" ran we: N01 prel cult non time oni prox of ti eiier elim ranged fi'om 25 to 30 times more (per mg mycelia) than in the shake cultures. The mycelia were collected from the remaining set of duplicate cultures and the RNA extracted. Northern hybridization analysis with nor-1 and ver-l probes showed that transcripts of the predicted size were present in the A. flaws producers at both time points in stationary cultures but lower levels were detected in the shake cultures. AS, A0, and the A. flavus non-producers did not produce detectable levels of transcript of the predicted size at either time point suggesting that nor-1 and ver-l are not expressed. Identifying how non- toxigenic species differ from toxigenic species in the regulation of aflatoxin genes may provide some clues about the origin of the aflatoxin biosynthetic pathway, and the causes of the loss of toxin production in non producers. Ultimately, regulatory factors which cfl‘ect aflatoxin synthesis may be identified and their production or activity manipulated to eliminate aflatoxin production. To Fred. iii cont rnadc ACKNOWLEDGMENTS I would like to take this opportunity to thank all of those people who have made contributions not only to the work that I have done in the lab but also to those who have made a difi‘erence in any aspect of my life leading up to this publication. iv LIST LIST LITE MAT] RESU DISCL APPE.\ APPE.\ TABLE OF CONTENTS LIST OF TABLES ................................................... vii LIST OF FIGURES .................................................. viii LITERATURE REVIEW ............................................... 1 Overview ...................................................... 1 The Genus Aspergillus ............................................ 2 Sources of Aflatoxin Production .................................... 6 Prevalence of Aflatoxin Producing Strains ............................. 8 Conditions for Fungal Growth and Aflatoxin Production .................. 9 Biological Effects of Aflatoxin ...................................... 9 Aflatoxicosis ....................................... 12 The Aflatoxin Health Hazard ...................................... 12 Methods of Controlling Aflatoxin Production .......................... 14 Short Term Goal ............................................... 17 MATERIALS AND METHODS ......................................... 19 Strains and Culture Conditions ..................................... 19 Extraction of Genomic DNA ...................................... 21 Southern Hybridization Analysis .................................... 21 Extraction of Aflatoxins from Mycelia ............................... 22 ELISA Analysis for Aflatoxins ..................................... 22 Extraction of Total RNA ................................ - ......... 22 Northern Hybridization Analysis .................................... 23 Thin Layer Chromatography ...................................... 23 Precautions When Handling Aflatoxins ............................... 24 RESULTS .......................................................... 25 DISCUSSION ....................................................... 45 APPENDIX A: Sequence analysis of the fas-lA, fas-ZA, and clone 6 ............. 51 APPENDIX B: Sequence analysis of the A. parasiticus pyrG ................... 93 APPENDIX C: Protocols used in promoter studies .......................... 100 LIST LIST OF REFERENCES ............................................. 107 LIST OF TABLES Page Some industrial Aspergillus species and the enzymes they produce (adapted from Smith, 1994) ........................................ 5 Sensitivity of Different Species to Aflatoxin Bl fed Orally (adapted from Ellis et a], 1991) ............................................ 10 Gene fragments used as probes in northern hybridization analysis ........... 23 Dry weights (grams) ofAspergillus spp. grown on Reddy medium to 48 and 72 hours in shake and stationary cultures. ...................... 29 Analysis of aflatoxin production from stationary cultures. ................. 30 Analysis of aflatoxin production from shake cultures. .................... 31 vii 14. LIST OF FIGURES figure has 1. Diagrams of Aspergillus ........................................... 3 2. Structures of four important toxins produced by species of Aspergillus ....... 6 3. Structure of the highly reactive epoxide form of aflatoxin B1. .............. 10 4. Southern hybridization of Aspergillus flavus strains with nor-1 and 10. 11. 12. 13. 14. 15. ver-l probes ................................................... 27 Southern hybridization of strains 1273, 2112, and SU—l with A) nor-1 and B) ver-l probes. ............................................ 28 Northern hybridization of A) shake cultures, B) stationary cultures, and C) A0 shake and stationary cultures using the B-tubulin probe ............. 34 Northern hybridization of A) shake cultures, B) stationary cultures, and C) A0 shake and stationary cultures using the pyrG probe ................ 36 Northern hybridization of A) shake cultures, B) stationary cultures, and C) A0 shake and stationary cultures using the nor-l probe ............... 41 Northern hybridization of A) shake cultures, B) stationary cultures, and C) A0 shake and stationary cultures using the ver-l probe ................ 43 Map of the Cosmid NorA ......................................... 54 Amino acid comparison of the suspected acetyl transferase domain of fax-1A ....................................................... 56 Amino acid comparison of the suspected dehydratase domain of fas-lA ...... 58 Amino acid comparison of the suspected acyl carrier protein domain of fas-2A ....................................................... 6O Amino acid comparison of the suspected B-ketoacyl reductase domain offas—ZA ..................................................... 62 Amino acid comparison of the suspected B-ketoacyl synthase domain offas-2A ..................................................... 65 viii 16. 17. 18. 19. 20. Nucleotide sequence of the fas-2A of A. parasiticus ..................... 68 Amino acid comparison of a suspected cytochrome P450 domain ........... 73 Nucleotide sequence of the cytochrome P450 like region of clone 6 from the Cosmid NorA ........................................... 75 Nucleotide sequence of the A. parasiticus pyrG ........................ 95 Amino acid comparison of the A. parasiticus pyrG to other genes encoding 0MP decarboxylase ..................................... 97 ix Ove fun: theil 199 mut I99 Emir afla OCC Eff: tha tha in c dIS( Literature Review Overview The aflatoxins are a group of secondary metabolites produced by the imperfect fungi Aspergillusflavus, A. parasiticus, and A. nomius. During the three decades since their initial discovery, 17 different aflatoxins have been identified (McLean and Dutton, 1995). Some of these chemicals have been found to be extremely carcinogenic, mutagenic, and teratogenic. Of these, aflatoxin B1 (AFBl) is the most potent (Ellis et a1, 1991) and, in fact, is the most potent naturally occurring carcinogen known (based on animal studies) (Diener et a1, 1987; CAST, 1979). Of the remaining 16 aflatoxins, aflatoxin Gl (AF G1) is the most toxic though it is far less reactive than AFBI. The occurrence of aflatoxins in nature is dependant on where the fungal strains can grow. In general, AFBl is more common than the other aflatoxins and again AF Gl comes in a distant second (Ellis et at, 1991). The aflatoxins pose a health threat to all animals tested thus far. Adverse health effects have been seen in rats, monkeys, turkeys, and trout (to name a few test species) that have been fed a diet containing aflatoxins. This evidence has led to the hypothesis that aflatoxins may be dangerous to humans as well. This threat is not a primary concern in developed countries where foods found to be contaminated by aflatoxins can be discarded, rather it is an economic concern due to wasted product. In non-developed cour pote the f stanc few) poun mani‘ The i Mich. Chara there fbnnl dISper Amer; Similar work 1965) 86nus, Vafieue morpho 2 countries food may be scarce, therefore, even if contaminated, the food is eaten causing a potential health problem. Due to these two problems the elimination of aflatoxins from the food chain is a desired goal. Several methods have been considered to achieve this goal. Among them, standard farming practices prior to harvest, and post harvest decontamination (to name a few) have been investigated and deemed unfeasible for reasons to be discussed later. One potential method being explored to eliminate aflatoxins is increasing host plant defenses by manipulating them genetically. The Genus Aspergillus P.A. Micheli first described species of Aspergillus in 1729 (Bossche et al, 1987; Micheli, 1729). He noted that the species of Aspergillus can be morphologically characterized by aerial hyphae rising out of septate mycelial cells. At the tip of each hypha there is a vesicle to which numerous sterigma are attached. Chains of conidia (spores) form from these sterigma (Figure 1a)(Bossche, 1987). The spores are cells which are dispersed via the air to allow propagation of each species. Micheli derived the name Aspergillus from the name aspergillum, a holy water sprinkler, due to the striking similarity in appearance (Figure 1b, c). Since Micheli’s description the genus has grown. Work by Raper and Fennel identified 132 species in 1965 (Smith, 1994; Raper and Fennel, 1965). In 1984 Christensen and Tuthill proposed a total of 276 species and varieties in the genus Aspergillus (Smith, 1994; Cristensen and Tuthill, 1984). These species and varieties have been placed into 18 groups (for fast characterization) based on morphological and cultural characteristics. Figure 1. Diagrams of Aspergillus. A) Diagram of conidiophore structure typically found in Aspergillus spp. B) First drawings of observed Aspergillus by Michelli. C) An aspergillum - a holy water sprinkler (Bossche, 1987). Figur H v\‘.‘.-.‘ ".1 .;/, \HWZ ~\“\\f “II/v ./ /. :- \V“l ’ ~\$\ ,\; \. \~\- ‘ '1‘ :1 u" t—~.hlclldo i. ‘ ‘1 Ionic-“J I! \—.———v..m.-—--I‘ 3‘.- _-_...-......u'._.- ....... '_ .: AsPergiILus 1:23-33:13. I108 @5191 II?“ T T -D n.1‘r .;.' L" «9‘..le .ci‘ €£_:\‘Ttt’:- -. 5 Many species within Aspergillus are of great importance to humans. Some species are beneficial. For example, some strains of A. sojae and A. oryzae have been used in the fermentation of shoyu (soy), miso, and sake for hundreds of years (Bossche, 1987; Smith, 1994). Also used in the food industry are enzymes such as rat—amylase, glucoamylase, and lipase which are produced by strains of A. niger, A. oryzae, and A. malleus respectively (Smith, 1994) (Table 1). Table 1. Some industrial Aspergillus species and the enzymes they produce (adapted from Smith, 1994) A. awamori A. japom'cus A. niger A. niger . niger . niger . niger Emma. . niger A. niger A. oryzae Aspergr’llus species Aspergillus species Glucose oxidase Glycerol oxidase Amyloglucosidase B-Glucosidase B-Galactosidase Catalase Lipase Metalloproteinase Pectinase a-Amylase D-Glucanase O-Glucose dehydrogenase By contrast, other species ofAspergillus are known to be harmfirl to humans. One way is the ability of some Aspergillus strains to cause pathogenic diseases in humans. The most common example of pathogenic Aspergillus is A. filmigatus (Bossche, 1987). Other specie A. par meet 2). E2 6 species are hannful in that they produce toxic compounds. Species such as A. ochraceus, A. parasiticus and A. flaws, and A. versicolor present problems in that they produce the mycotoxins ochratoxin A, aflatoxins B1 and G1, and sterigrnatocystin respectively (Figure 2). Each of these toxins has been hypothesized to be toxic to humans (Bossche, 1987). Afiatoxin G1 Sterigmatocystin Figure 2. Structures of four important toxins produced by species of Aspergillus Sources of Aflatoxin Production The aflatoxins were initially identified in the 1960's as the causative agent of a deadly epidemic of turkey polts. This disease, known as Turkey “X” disease, claimed the lives of more than 100,000 turkeys. The vehicle of intoxication was found to be A. flavus contaminated feed (Ellis, 1991; Buchi and Rae, 1969). It was later discovered that aflatoxins are metabolic products of certain strains of A. flavus. Since then it has been 7 shown that all known strains of A. parasiticus, with one exception, also produce aflatoxins (CAST, 1989). A. flaws and A. parasiticus were generally accepted as the only aflatoxin producers until recently. A report by Kurtzman described a third species capable of producing aflatoxins-A. nomius (Kurtzman et a1, 1987). A. flaws spores are generally smooth, thin walled, and variable in shape - ranging fi'om spherical to ellipsoid (Pitt, 1993). According to the ATCC, 29 isolates of A. flavus produce aflatoxins. It has also been reported that all of these isolates produce the B aflatoxins (ATCC, 1991). Six isolates (of the 144 reported in the 1991 ATCC catalogue) are reported to produce aflatoxins G1 and G2 as well. Strains of this species also produce toxins other than aflatoxins. One of the most important of these toxins, with respect to the food industry, is cyclopiazonic acid. The importance of cyclopiazonic acid is not due to its potency (which pales in comparison to that of aflatoxin) rather it stems from the fact that large quantities are produced after the fungus has colonized food products. Some strains of A. flavus have even been described in pathogenic infections causing acute aspergillosis (Bossche, 1987) in immunocompromised persons. The strains of A. parasiticus difl‘er morphologically from those of A. flavus in that A. parasiticus spores have thick, rough walls that are uniformly spherical (Pitt, 1993). There are fewer reported isolates (39), and all but one of the strains produce aflatoxins. The exception is a strain that produces o-methylsterigmatosystin (ATCC, 1991), a toxic precursor of aflatoxin. These strains produce B aflatoxins (B1 and Bl) as well as the G aflatoxins (G1 and G1) - all four major aflatoxins. A. parasiticus also produces aspergillic acids and kojic acid which are not considered to be important toxins in the food industry. A search of the Medline, Agris, and Toxline literature databases did not indicate that A. 8 parasiticus has been implicated in pathogenic infections. A. nomius is very difficult to distinguish from A. flavus because it too produces spores with smooth, thin walls of variable shape (Pitt, 1993). This species produces sclerotia which is the only morphological feature used to separate it from A. flavus (Pitt, 1992). A. nomius, like A. parasiticus, is able to produce the B and G aflatoxins and can serve as a fair indicator to distinguish A. nomius from A. flavus. An indirect source of aflatoxins (via foods) is dairy cows. The use of aflatoxin contaminated crops as feed for cows results in the metabolism of aflatoxin Ml (a hydroxylated form of AFB.) by cows. This aflatoxin is found in the milk and meat that the cow produces. This contamination poses obvious potential health risks to human, most notably to infants consuming milk. Prevalence of Afiatoxin Producing Strains A. flavus, A. parasiticus, and A. nomius are considered ubiquitous in nature. That is they grow in many different environmental conditions and on various substrates. This is due to the fact that they are typical fungi and, therefore can grow over wide ranges of pH, temperature, and water activities (Cotty et al, 1994). The versatility of these aflatoxin producing fungi make them dangerous because they are able to grow on many food commodities. These commodities include cotton, cottonseed, corn, and peanuts. Growth of these fungi on these crops is opportunistic. This means that growth occurs on plants that are damaged in some way. Corn, for example, may be damaged before (by insects), during (by farming equipment), or after (handling practices/storage) harvest. As few as one damaged kernel can allow colonization by Aspergillus (Cotty et al, 1994). Subs proc: ornr aflau Conc condh Aflau: impor rdauv 1979), Biolog 0n anir Species VaDalio and difl Collier-[E me‘hOd . 9 Subsequent dispersion of spores can occur at any time from growth in the field through processing, thereby increasing the potential for contamination. The colonizing fungus may or may not be an aflatoxin producer (as some strains of A. flaws do not produce aflatoxins) but the presence of the fungus can be sufficient for rejection of the crop. Conditions for Fungal Growth and Aflatoxin Production In general, strains of A. flaws and A. parasiticus grow optimally under similar conditions. The optimum grth temperature is between approximately 25 °C and 30°C. Aflatoxin production takes place optimally around 29 to 30.5 °C. The second most important growth factor is the relative humidity. For A. flaws and A. parasiticus a relative humidity between 88 and 95% results in optimal growth (Ellis, 1991; Bullerrnan, 1979) Biological Effects of Aflatoxins Many studies have been undertaken to determine the effects of aflatoxin poisoning on animals. It has been noted that while there are vast differences in susceptibility from species to species, those tested thus far are adversely affected by aflatoxins (Table2). The variations seen are caused by many factors including age, sex, nutritional status, health, and differences in activation and detoxification systems in the hosts cells. Aflatoxins in their native form have low toxicity. After ingestion they are converted (activated) to a more toxic form and begin to exert their effects. The major method of conversion is by cytochrome P450 binding of an aflatoxin molecule (Massey et al, 1995; Gurtoo and Dave, 1975; Essigmann et al, 1982). This binding leads to the toxic 10 form of AFB, via conversion of the double bond in the furan ring to an epoxide (Figure 3). Table 2. Sensitivity of Different Species to Aflatoxin B, fed Orally (adapted from Ellis et al, 1991) Species LDso (mg/kg body weight) Rabbit 0.3 Cat 0.55 Rainbow Trout 0.8 Dog 0.5 - 1.0 Guinea Pig 1.4 - 2.0 Baboon 2.0 Chicken 6.3 Rat (male) 5.5 - 7.2 Rat (female) 17.9 Mouse 9.0 AFB1 8.9-epoxide Figure 3. Structure of the highly reactive epoxide form of aflatoxin B,. The 8,9-epoxide is highly reactive and capable of covalently binding to macromolecules such as DNA and proteins (via the hydroxylated AFB”) (Eaton and Groopman, 1994; Dvorackova, 1990). 11 In the case of DNA binding, the reactive aflatoxin molecule shows an affinity for binding to the NI position of guanine bases (Massey et a1, 1995; Harrison and Garner, 1991; Ball et al, 1990). The binding of DNA is postulated to be responsible for the mutagenic and carcinogenic effects of aflatoxins. Two methods of mutation exist after binding to DNA. The first method is disruption of replication and/or transcription due to the altered structure of the DNA (Hsieh, 1987). It is clear that disruption of binding by or processivity of polymerases (both DNA and RNA) are major concerns caused by structural alterations. The second method is that AFB,-DNA adducts formed can result in GC-DTA transversions during replication (Croy and Wogan, 1981). These transversions may lead to inactive protein molecules and possibly death of the cell depending on the importance of the altered gene. Another fate caused by transversion is the mutation of the p53 tumor suppressor gene. Studies have shown that the carcinogenic effects of aflatoxins result from a specific mutation of codon 249 of the p53 tumor suppressor gene (Massey, 1995; Bressac et al, 1991; Hsu et a1, 1991). Protein binding by aflatoxins also poses two threats. The most obvious of the two is that binding can cairse conformational changes of proteins. By disrupting the folding of an enzyme the catalytic site of that enzyme may lose some or all of its activity. The second threat is the binding of aflatoxins by proteins which carry them to the nucleus. Obviously, this increases the possibilities of DNA binding (Massey et al, 1995). Removal of the reactive epoxide form of aflatoxin is accomplished predominately by a glutathione S-transferase (GST) system. This system can be found in the cytosol and microsomes. GST catalyzes the conjugation of activated aflatoxins with reduced glutathione. This conjugation leads to the excretion of the activated aflatoxin (Neal and Green. The a system confer A flator broken 0f aflal and int Where: chroni Pfimal SOmet manr leads- 3150 1, note 1 probli mEljor 12 Green, 1983). The GSTs are composed of alpha, mu, pi, theta and microsomal classes. The activity of each class varies fiom species-to-species. Variation in activity of the GST system combined with the variation in activity of the cytochrome P450 activation system confer species differences in susceptibility to aflatoxins. Aflatoxicosis The disease caused by aflatoxin intoxication is aflatoxicosis. This disease can be broken down into three types based on its severity which is often determined by the levels of aflatoxins consumed. Acute and chronic aflatoxicoses result from consumption of high and intermediate levels of aflatoxins respectively. Acute aflatoxicosis results in death whereas chronic results in a reduction of growth rate and reproduction. Acute and chronic aflatoxicoses are considered subdivisions of primary aflatoxicosis. Both of the primary aflatoxicoses can be characterized by adverse effects seen in the liver, lungs, and sometimes the kidneys. Secondary aflatoxicosis results from low levels of aflatoxins. The major symptom of secondary aflatoxicosis is the weakening of the immune system. This leads to the inability of animals to defend themselves against invasion by pathogens and also hinders attempts at vaccination (Ellis et al, 1991; Piers et a1, 1979). It is interesting to note that the effects of secondary aflatoxicosis may be cumulative. This would present the problem that no level of aflatoxin consumption is acceptable and that aflatoxins are a major health concern not only in third world countries but also developed countries. The Aflatoxin Health Hazard A major controversy has developed with respect to whether or not aflatoxins are, in fact, a health hazard to humans. The origins of this controversy can be identified as the l3 1) differences in susceptibility from one test animal to the next and 2) the high incidence of other health risks in areas along with aflatoxins. Obviously, no direct experiments have been carried out using human test subjects, leaving this question to be resolved with data from test animals. Differences between activation-detoxification systems have led to variable data from species to species (though the data do indicate that all test animals have shown adverse efl‘ects). Because of these difl‘erences it has been difficult to extrapolate the possible efl‘ects aflatoxins may have on humans. Outbreaks of liver cancer involving human populations, however, have helped to substantiate the circumstantial evidence obtained from test animals. Major outbreaks of hepatic cancer believed to be caused by aflatoxin consumption have occurred in India (Hsieh, 1986), China (Bressac et al, 1991), and Africa (Hsu et al, 1991). Opposing these data is the belief that primary liver cancer is the result of infection by hepatitis B virus (HBV) which is present in areas of high incidence of aflatoxin consumption (Stoloff, 1989). Tests have shown that there are two major organs affected by aflatoxins: the liver, and the lungs. The liver is the site of the major AFB, activation system (cytochrome P450) and, therefore is the major organ affected by aflatoxin poisoning. Poisoning of test animals’ livers has resulted in hepatic cancer. An alternate route leading to lung aflatoxin exposure is via inhalation of dust and grains infested with aflatoxins which poses a threat to farm workers. Symptoms of aflatoxin poisoning of the lungs include cancerous growths and congestion (Moreau and Moss, 1979). The effects of aflatoxins on the lungs were shown following an incident in which several persons in Southeastern Asia died after consuming a diet containing aflatoxins. Aflatoxin-DNA adducts were revealed in the lung tissue of these victims 14 (Harrison and Garner, 1991). Other studies, using human cell lines, demonstrated the ability of lung cells to activate AFB, in the same manner as liver cells (cytochrome P450) (Autrup et al, 1979; Stoner et al, 1982; Manda], 1987). Methods of Controlling Aflatoxin Production Many methods for controlling aflatoxins have been investigated. Among these methods are standard farming practices, biocompetition, and increased host defense mechanisms. Standard farming techniques entail thorough irrigation, carefirl harvesting practices and special environmental storage. In areas of persistant drought, adequate irrigation, let alone thorough, may be impossible. Even in countries where drought is not a problem thorough irrigation is deemed unfeasible due to the high cost of such a practice. Aside from the availability and cost problems of thorough irrigation there is the needs of different crops for different grth conditions. For example, the final stage (ripening) of peanut grth is characterized by drought stress. Obviously, irrigation of peanut crops during this stage would be detrimental to the final product. Carefirl harvesting practices are designed to reduce contamination by reducing the damage to and, consequently the susceptibility of crops. These practices do not ensure a reduction of contamination because it does not account for the damage of crops by insects. Another problem with carefirl handling practices is the cost of implementing them. This would be the result of slower harvesting and/or increased number of workers. Special environmental storage conditions describe the method of storing crops in silos that have controlled humidity and temperature. This method is capable of preventing both grth of and toxin production by fungi. The major problem with this method is once again money. To set up and 15 operate such a system costs far more than it may be worth. Also, this method is not able to stop grth by all strains at all times because of the ubiquity of the strains. Biocompetition is a control method that has received much attention in recent years. This method involves seeding the soil of a field with a non-toxigenic strain early in the growing season. The hope is that this non-toxigenic strain will grow faster than the toxigenic strains thus excluding their growth. This method does not propose to introduce more fungal growth in the field, it simply aims to replace growth of toxigenic fungi with non-toxigenic fungi. Studies have shown (Cotty et al, 1990; Horn et al, 1994; Ehrlich, 1987) that this effect can be seen when applied to test plots. Domer et al (1992) reported that the application of a competitive strain can decrease the total growth of other strains and total production of toxin. This “inhibitory” effect increased for as many as four years. At least two problems exist with this method of control. The first problem is the selection of strains for particular farming regions. The fungi that produce aflatoxins are ubiquitous in nature. Each strain, however, is suited to grow optimally in a particular set of environmental conditions (climate). This means that a strain which grows better than others in one climate may not be able to grow better than those same strains in another climate. In fact, it may be entirely possible that that same strain will not be able to grow at all in the second climate. In relation to this problem, fluctuations in climate (within a region) must be considered when selecting a biocompetitive strain. Changes in climate from year-to-year within the same region may have the same affect as described for differences between regions having different climates. Selection of a strain may not be a problem if the field is seeded with high numbers of the control strain at an early enough time in the growing season. The second problem encountered when using this method is 16 the fact that it does not reduce the contamination of crops by firngal growth. Inherently, this method requires contamination. Because a crop can be rejected on the basis of fungal contamination this method does not eliminate one of the main concerns generally ascribed to aflatoxin production - economic. Increased host defense mechanisms refers to the ability of a plant to defend itself against colonization and/or production of toxin by fimgi. Chemicals have been identified that inhibit growth and/or toxin production. By use of molecular techniques the effects of inhibitory chemicals can be characterized at a genetic level. These methods may show that an inhibitor acts at the biochemical pathway which is responsible for the production of aflatoxins. Alternatively, they could show that an inhibitor may act as a firngicide thus preventing grth of the fungus. If the production of these chemicals can be incorporated as a host plant defense mechanism then it is possible that the colonization by or production of toxin can be reduced. Problems that exist with this strategy are the identification of inhibitors, the number of possible crops that would need modification, and the reception of this method by the public. The first problem can be addressed by trying to find inhibitors produced by the crop itself - which would be preferred. For example, peanuts produce 5,7-dimethoxyisoflavone. When extracts of this compound are applied to developing plants in the presence of toxigenic A. flavus spores grth of the fungus is greatly reduced (Turner et al, 1975). Increasing the production of this inhibitor by modifying its regulation may adequately protect peanut crops. The second problem may be resolved by the possible solution of the first. If each crop naturally produces an inhibitor of either growth or toxin production, the modification of each crop may be similar for many of the crops. The final problem is typically defined as a mistrust of genetics by the public. Past attempts 17 to market products that have been associated with genetic modification, such as the fi'ost free strawberry, have met much resistance. A way around this problem may be breeding natural producers of inhibitors to express those inhibitors at higher levels, this solution is dependent on the resolution of problems one and two. Nevertheless, the marketing of such products will be highly dependant on education of the non-scientific community. This education must start with regulators to ensure that reasonable (as opposed to excessive) precautions/warnings are assigned to these products. In this way a paranoia by the general public can be avoided to some extent. Ultimately, the public will decide on the feasibility of modified products. It is easy to confuse persons on complicated issues. Terms such as “antisense RNA” and “gene inactivation by deletion” are not likely to become household jargon any time soon. People will always be able to introduce some doubt, confusion or even fear of these kinds of products. The advent of the flavor savor tomato was a step in the right direction but to continue that progress a spokesperson or committee aimed at educating the public about new products should be formed. Short Term Goal Clearly, the long term goal of aflatoxin research is to eliminate aflatoxins from the food chain. The work presented in this paper takes a molecular approach toward achieving this goal. The main focus of this work was to characterize growth, aflatoxin production, and the presence of aflatoxin genes and transcripts by and in several species of Aspergillus. The strains selected for this study include one A. parasiticus, two A. flavus aflatoxin producers, two A. flavus suspected aflatoxin non-producers, one A. sojae suspected aflatoxin non-producer, and one A. oryzae suspected aflatoxin non-producer. 18 At the beginning of this project, it was hoped that characterizing the differences between these strains might lead to clues as to why some strains produce aflatoxins whereas others do not (even if they have the aflatoxin genes). With this kind of information it may be possible to genetically modify toxigenic strains in such a way that they behave like non- toxigenic strains. It was also hoped that we could determine if industrial strains (represented by A. sojae and A. oryzae) also possess the biosynthetic pathway for aflatoxin production. At the moment it is unknown why some strains produce aflatoxins whereas others do not (this referring to A. flavus more than A. parasiticus or A. nomius). It is thought that the inability of some strains to produce aflatoxins is brought about by mutations to regulators of the pathway rather than enzymes of the pathway. Several studies have shown that disrupting enzymes results in an accumulation of toxic aflatoxin precursors. Disruption of genes such as nor-1 (Trail et al, 1994), ver-l (Skory et al, 1992), and amt-1 (Yu et al, 1993) result in an accumulation of norsolorinic acid, versicolorin A, and sterigrnatocystin, respectively. Three genes afl-R (Payne et al, 1993), fax-1A (Mahanti et al, 1996), and pksA (Chang et al, 1995) have been disrupted without an accumulation of precursors. It has been hypothesized that the afl-R is a regulator though its mechanism of action has not been shown. 19 Materials and Methods Strains and Culture Conditions Growth conditions for dry weight, aflatoxin and RNA extraction Seven strains ofAspergilIus were used in this study. A. parasiticus NRRL 5862 (SU-l), A. flaws SRRC 1059 (1059), and A. flavus SRRC 1273 (1273) comprised the aflatoxin producing strains. A. flavus SRRC 2112 (2112), A. flavus SRRC 2115 (2115), A. oryzae ATCC 14895 (A0), and A. sojae ATCC 42251 (AS) were the suspected aflatoxin non-producers. Cultures of each strain were grown in duplicate by inoculating 100ml ofReddy’s medium (in 250ml flasks)(Reddy et al, 1971) with 107 spores per flask. Each pair of cultures was allowed to grow in the dark at 29°C while stationary or shaking at 150 rpm (in a New Brunswick G-25 floor model shaker). Two time periods of growth were selected, 48 and 72 hours, based on the fact that the aflatoxin gene transcripts are not easily detectable before 48 hours and begin to degrade at some time between 72 and 84 hours (Skory et al, 1993). It should be noted that the aflatoxin gene transcripts may be produced earlier than 48 hours but insufficient cell mass (for RNA extraction) was available before that time in the stationary cultures. At the end of the grth period the contents of the flasks were vacuum filtered using a Buchner funnel and mira cloth. For every flask, five ml of growth medium were removed and stored at -20°C prior to ELISA analysis for aflatoxins. From the first flask of each pair the mycelia were scraped from the mira cloth and dried under vacuum at 80°C for one hour. The mycelia from the second 20 flask were also scraped ofi‘ the mira cloth but this material was halved for RNA extraction and aflatoxin extraction (for ELISA analysis). Growth conditions for DNA extraction Approximately 107 spores were inoculated into 100ml of yeast extract (20g/l)-sucrose (60g/l) medium (YES) in a 250ml flask. The flasks were incubated for 48 hours in the dark at 29°C while shaking at 150 rpm. Spore preparation procedure One hundred fifty ml of Potato Dextrose Agar (PDA) were solidified in a one liter Brockway glass bottle and inoculated by streaking spores across the agar surface with a sterile loop. The cultures were allowed to grow for 10 days at 29°C in darkness. The spores were harvested by aseptically pouring approximately 50 - 100ml of sterile water into the bottle followed by the addition of a sterile magnetic stir bar (Baxter). The bottle was then placed on a NuovaII stir plate to remove the growth from the agar. The contents (excluding the stir bar) of the bottle were poured into a 60m] Tuberculin syringe (Becton Dickinson & Co.) containing a plug of sterile glass wool (Pyrex). The growth suspension was allowed to filter through the syringe and glass wool (the spores passed through the glass wool but the mycelial fragments were retained) into a 250ml GSA bottle (Sorvall). At the same time another 50 ml of sterile water was added to the bottle to remove any growth that may still have been in the bottle. This suspension was also added to the syringe. The contents of the syringe were then compressed with the plunger to remove as much liquid (presumably containing spores) as possible. The GSA bottle was then centrifuged in a RC2 (Sorvall) centrifuge at 5856xg for 10 minutes at 4°C. The supernatant was carefully poured off and the pellet was suspended in approximately five 21 ml of sterile 20% glycerol (Boehringer Mannheim Biochemical). Ten pl of the sample was used to determine the concentration of the suspension using a hemocytometer and bright field microscope (American Optical). The spore suspension was finally distributed to 1.5ml micro centrifuge tubes and stored at -80°C until used. Extraction of Genomic DNA Extraction of genomic DNA was performed by grinding mycelia in a mortar using a pestle and liquid nitrogen, followed by two phenol-chloroform extractions and ethanol precipitation (Ausubel et al, 1987; Skory, 1992). Southern Hybridization Analysis Genomic DNA was cut with restriction enzymes (Boehringer Mannheim Biochemical) and electrophoresed on a 1% agarose gel for four hours at 80 volts. Following electrophoresis the DNA was transferred to Nytran (S&S) nylon membrane using the method of Maniatis (1989). Southern hybridization analysis was performed at 42°C for 16 hours (in a Robbins Hybridization Incubator 310) using the labeled (”P) nor- 1 (1 .5-kb EcoRI/CIaI fi'agment of pNA17) and ver-lA (1.8 -kb EcoRI/SalI or 0.7-kb EcoRI/BamI-II fragment of pBSV2) genes from A. parasiticus NRRL 5862 as probes. The membranes were washed twice for 15 minutes with 2X8 SC, 0.1%SDS at room temperature followed by a one hour wash at 65 °C using a 0.1XSSC, 0.1% SDS buffer. Autoradiography was performed with Kodak XAR-S film at -80°C. 22 Extraction of Aflatoxins from Mycelia Aflatoxins were extracted from mycelia by adding five ml of 100% methanol to half the mycelial contents of one growth flask. The methanol-mycelia samples were agitated briefly and then allowed to stand for at least one hour prior to removing the methanol which was then stored at -20°C until needed for thin layer chromatography (TLC) or ELISA analysis for aflatoxins (Dvorackova, 1990). ELISA Analysis for Aflatoxins Direct competitive ELISA analysis was performed by the method of Pestka (1988). The polyclonal antibody, 5C11, (directed against AFB ,) and the aflatoxin- peroxidase conjugate, 80B, were kindly provided by Dr. James J. Pestka. A 1:500 dilution of the antibody as well as the aflatoxin-peroxidase conjugate were used. Aflatoxin standards were prepared with an aflatoxin B,, B2 mixture (Sigma Chemical Co., St. Louis, MO) which was dissolved in 100% methanol. Some of this stock was diluted to one ng/ul for preparation of the standards (0.5, 1.0, 5.0, 10, 50, and 100 ng/ml) used in the procedure. The absorbance of each well of the microtiter plates was read using a Vmax spectrophotometer (Molecular Devices) using Softmax Software running on an IBM model Z50 computer. Extraction of Total RNA Total RNA was extracted fiom approximately 1.5 g of wet mycelia (if available) by the hot phenol method of Maramatsu (1973). 23 Northern Hybridization Analysis Forty ug of total RNA were electrophoresed on a denaturing formaldehyde agarose gel (0.8%) at 75 volts for 3 .5 hours. The RNA was then transferred to Nytran plus nylon membrane (S&S) by the method of Maniatis (1989). The membranes were then probed for 16 hours at 42°C using the labeled (32F) DNA fragments in Table 3. The membranes were then washed. Autoradiography was performed with Kodak XAR-S film at -80°C. Table 3. Gene fiagrnents used in Northern Hybridization analysis Gene Plasmid Restriction enzyme(s) Size of fiagment a -R SacI fragment EcoRI 0.5 -kb B-tubulin pAPBENK SacI - KpnI 1.0 -kb nor-l pNA-17 HindIII - ClaI 1.0 -kb pksA pAPNVES4.3 EcoRI - SacI 4.3 -kb pyrG pPG3J PstI 1.2 -kb ver-l 2.1-kbver BamHI - EcoRI 0.7 -kb Thin Layer Chromatography (TLC) Thin layer chromatography was carried out to resolve aflatoxins for qualitative purposes. Whatman Silica plates (cat#05713264) were spotted with lO,uL of sample and 250ng of aflatoxin B, standard (Sigma). The plates were then placed in a chamber containing chloroform (1T. Baker) and acetone (Mallinckrodt) at a ratio of 95:5. The samples were resolved for 45 minutes. The plates were removed and allowed to dry, and then viewed under long wave UV light. The appearance of blue bands which comigrated 24 with standard indicated the presence aflatoxin B ,. Precautions When Handling Aflatoxins Because aflatoxins are potent carcinogens the handling and disposal of them are must be carefully executed to avoid exposure. Whenever a container with aflatoxins (whether in liquids or cells) was handled a laboratory coat and gloves were worn. Grinding of mycelia (during DNA and RNA extractions) results in particulate “mycelial” dust, therefore, this procedure was carried out in a biological hood and a mask was worn. Disposal of aflatoxins involved chemical inactivation. This was accomplished by treating all aflatoxin contaminated containers, grth media, and cells with a 5% bleach solution (final concentration) for at least a half hour. Acetone was then added to a concentration of 5% to stabilize the reaction. 25 Results Southern hybridization analysis Southern hybridization analysis using high stringency washing conditions was conducted on genomic DNA extracted from A. parasiticus SU-l (wild type) by Skory (1992). Several restriction enzymes were used to cut the DNA before being probed with (”P) ver-lA and (”P) nor-l DNA probes. Nine of the enzymes used (known not to cut within the ver-lA) produced two DNA fragments capable of hybridizing the ver-lA DNA probe. The BamI-II digest produced four bands even though it was known only to out once within ver-lA. These data led to the identification of a second copy of the ver-lA gene in A. parasiticus - called ver-lB. Nucleotide sequence analysis of ver-lB demonstrated that it is 93% identical to the ver-IA at the nucleotide level (Liang et al, 1994). Furthermore, a translational stop codon was reported early in the ver-lB coding sequence prompting the belief that this gene produces a truncated protein with little or no function. Hybridization of these same digests with the nor-1 probe indicated that only one copy of nor-l is present in A. parasiticus based on the fact that no unexpected bands were identified (Skory, 1992). Southern hybridization analysis using was conducted on BamHI digested genomic DNA of several Aspergillus spp. (both aflatoxin producers and aflatoxin non-producers) in order to determine if genes similar to ver-lA and nor-1 could be identified in these species. Only A. versicolor and A. tamarii failed to hybridize with either probe whereas A. nidulans hybridized with the ver-lA probe but not the nor-1 probe under high stringency 26 conditions. For the rest of the species investigated the probes hybridized to only one DNA fragment each suggesting that only one copy of a gene similar to ver-lA and one similar to nor-1 is present (Skory, 1992). These data suggested that the aflatoxin genes are not present soley in aflatoxin producing strains. The aflatoxin non-producing strains included three A. sojae and one A. oryzae. These findings are of particular interest due to the industrial use of some strains of these species in fermentations. Rayard Thomas, in our laboratory, conducted further Southern hybridization analyses on HindIII restricted genomic DNA isolated from the following A. flavus strains: SRRC 284 (afl'), SRRC 285 (afl'), SRRC 2112 (afl‘), SRRC 2115 (afl‘), SRRC 1000A (afli), SRRC 1059 (afl+), and SRRC 1273 (afli). Using a 0.7-kb (EcoRI-BamI-II)ver-1A probe, Thomas was able to show that all but one of the strains contain a single ver-lA-like sequence of approximately 15.4-kb in size (unpublished data). SRRC 2112, however, produced two bands of equal intensity corresponding to approximately 15.8 and 13.8-kb (Figure 4). These findings suggested that 2112 (like SU-l) may contain two copies Of the ver-lA like sequence. One theory as to why nearly all A. parasiticus strains produce aflatoxins is due to duplications of genes in the A. parasiticus aflatoxin pathway. In theory, this would make A. parasiticus more resistant to mutations than A. flavus due to redundancy. SRRC 2112 is a suspected aflatoxin non-producer. If it does have multiple copies of the ver-lA sequence this may suggest that non-producers have gene duplications similar to the A. parasiticus strains. If this is so, it would be more likely that a mutation to a regulator has caused the inactivation of the aflatoxin pathway rather than mutations in a number of the genes. A. parasiticus has multiple copies of the afl-R (Payne et al, 1993) meaning that mutations to both copies would need to occur to eliminate its function. To 27 determine if suspected aflatoxin non-producers are deficient in the afl-R product it would be helpfill to determine the number of copies present in the genome. Using a 1.7 -kb (BglII—Sph1)nor-1 probe two major DNA fragments were detected in all A. flaws strains tested (Thomas). These fragments were approximately 3 .4 and 3 .2- kb in size (Figure 4). Additional weaker signals hybridized to the nor-1 probe in genomic DNA of strains SRRC 284, 2112, 1000A, and 1059 at 5.4 and 6.8-kb. Finally, 2112 genomic DNA hybridized to the nor-l probe also at 9.4-kb and 4.1-kb. These results indicate that there is one gene in all strains which appears to be nearly identical to nor-1 from SU-l. However, a variable number of less related genes may be present in strains SRRC 284, 2112, 1000A, and 1059. VER-TA NOR-T Figure 4. Southern hybridization of Aspergillus flavus strains with nor-l and ver-l probes. Southern analysis was performed once on SRRC 284, 285, 2112 and 2115 (aflatoxin non-producers); and SRRCIOOOA, 1059, and 1273 (aflatoxin producers). 28 Due to the fact that Skory and Thomas both used incompletely cut (HindIII) 127 3 genomic DNA samples when performing their Southern analyses it was necessary to perform another Southern analysis of this strain. The genomic DNA for 1273, 2112, and SU-l was prepared and fractionated as described in the materials and methods. Using the ver-lA and nor-l probes (listed in Table 3) it was found that only a single signal was present for each probe in all three strains (Figure 5). For the ver-lA probe this DNA fragment was significantly smaller (approximately 10. S-kb) than that observed by Skory and Thomas. Because the signals observed for 1273 and SU-l were the same size, however, the differences between these sizes and those seen by Skory and Thomas may be due to differnces in electrophoresis conditions and/or the transfer procedure. '5 <9 ,5" 4" ,5" o ~ x Q9 95’ 5 <29 45’ :~ 63’ (3' 699* eq' 65 s 23.1 ’ V 6.5 4.3 V ‘ 2.3 - , 2.0 ’ c Figure 5. Southern hybridization of 1273, 2112, and SU-l with A) nor-l and B) ver-l probes. 29 The nor-1 probe revealed one signal at approximately 3 .9-kb which is similar to the sizes reported by Skory and Thomas. The reason that there are two bands reported by Skory and Thomas is that their probes contained a HindIII site, whereas the nor-1 probe used to generate these data did not. Dry weight measurements Dry weights of the mycelia from each of the strains were determined in order to calculate the amounts of aflatoxins produced per milligram of mycelium (Table 4). The growth of the stationary cultures was very slow with total cell mass never reaching one gram. The shake cultures showed higher growth rates compared to those reported by Trail et al (1995). Table 4. Dry mycelial weights (grams) of Aspergillus spp. grown on Reddy medium to 48 and 72 hours in shake and stationary cultures. Weights are based on half a mycelial pad from one of the replicate flasks. Stationary F Shake 48 hours 72 hours 48 hours 72 hours SU-l 0.10g 0.12g 1.62g 2.19g 2112 0.15g 0.10g 1.11g 1.69g 2115 0.24g 0.14g 1.28g 1.58g 1059 0.21g 0.36g 2.21g 2.44g 1273 0.15g 0.28g 2.33g 2.78g AO 0.22g 0.19g 1.16g 2.25g AS 0.10g 0.11g 1.48g 2.53g The decreased growth in stationary cultures (compared to shake cultures) may be due to 30 decreased exposure to oxygen during germination and subsequent growth. Preliminary data suggest that the effect is caused by decreased germination rate. Zhou (personal communication) has indicated that shaking a culture for 24 hours then allowing it to grow as a stationary culture results in increased growth at later time points. The 24 hour time point is significant in his study because there does not appear to be any noticeable growth at that time. ELISA analysis ELISA was performed in order to determine the amounts of aflatoxins per milligram of mycelial dry weight being produced by the seven Aspergillus strains and the proportion of toxin production from mycelia and medium for the stationary and shake cultures respectively (Tables 5 and 6). A comparison of toxin levels found in the grth medium demonstrated that toxin production was generally reproducible in duplicate flasks. No aflatoxins were found in the media nor the mycelia from any of the suspected aflatoxin non-producers. Table 5. Analysis of aflatoxin B, production from stationary cultures. Replicates 1 and 2 indicate aflatoxin levels found in the media of replicate grth flasks. Only one extraction of mycelia was performed (therefore no replicate is Shown). Stationary 48 hours: Toxin in Toxin in Toxin in Total _ Dry Toxin/mg Replicate 1 Replicate 2 Mycelia Toxin Weight Mycelia SU-l 86.2ug 90.6ug 5.0ug 93.4ug 0.10g 930ng/mg 21 12 ND ND ND - 0. 15g - 21 15 ND ND ND - 0.24 g - 31 Table 5 (cont’d) AS ND ND ND - 0.10g - AO ND ND ND - 0.22g - 1059 101.9ug 75.6ug 5.2ug 93.9ug 0.2 lg 440ng/mg 1273 94.0ug 91.7ug 6.8ug 99.7ug 0. 15 g 660nymg Stationary 72 hours: SU-l 80.6ug 72.4ug 12.7ug 89.2ug 0.12g 740ng/mg 2112 ND ND ND - 0.10g - 2115 ND ND ND - 0.14g - AS ND ND ND - 0.1 lg - AO ND ND ND - 0.19g - 1059 57.4ug 94.0ug 21.1ug 96.8ug 0.36g 270ng/mg 1273 79.7ug 60.5 pg 9.99ug 80.1ug 0.28g 290ng/mg ND - none detected Table 6. Analysis of aflatoxin B, production from shake cultures. Replicates 1 and 2 indicate aflatoxin levels found in the media of replicate growth flasks. Only one extraction of mycelia was performed (therefore no replicate is shown). Shake 48 hours: Toxin in Toxin in Toxin in Total Dry Toxin/mg Replicate 1 Replicate 2 Mycelia Toxin Weight Mycelia SU-l 56.0ug 52.7ug 4.50ug 58.9ug 1.62g 36ng/mg 2112 ND ND ND - 1.1 lg - 2115 ND ND ND - 1.28g - AS ND ND ND - 1.48g - A0 ND ND ND - 1.16g - 1059 53.4ug 46.8ug 5.52ug 55.6ug 2.21 g 25ng/mg 1273 56.0ug 46.5ug 5.52ug 56.8ug 2.33g 24ng/mg 32 Table 6 (cont’d). Shake 72 hours: SU-l 121.9ug 123.8ug 12.5ug l35ug 2.19g 62ng/mg 2112 ND ND ND - 1.69g - 2115 ND ND ND - 1.58g - AS ND ND ND - 2.53g - A0 ND ND ND - 2.25g - 1059 110.0ug 128.2ug 12.6ug l32ug 2.44g 54ng/mg 1273 131.1ug 121.3ug 13.0ug l39ug 2.78g 50ng/mg ND - none detected The 48 and 72 hour stationary cultures produced roughly the same amount of AFB, which was nearly twice the amount produced by the 48 hour shake culture. The 72 hour shake culture, however, produced nearly 30% more AFB, than the 48 or 72 hour stationary cultures. In terms of AFB, production per milligram of dry weight, SU-l produced more toxin than 1059 and 1273 at either time point in stationary or shake culture. For example, SU-l produced 13% more toxin than 1059 at the 72 hour time point in shake. The equivalent stationary culture comparison revealed a difference of 64%. It can also be seen that the stationary cultures produced more toxin per milligram of dry weight than the shake cultures. For example, in the case of SU-l at 48 hours, the stationary culture produced nearly 30 times more toxin per milligram of mycelia. Because stationary cultures better mimic the conditions the fimgi are likely to encounter in the field, these cultures may give a more accurate account of aflatoxin production than shake cultures. If aflatoxin production is regulated at the level of transcription (Skory, 1993) then using stationary cultures may be more appropriate for studies of regulation. Finally, it appears that the mycelia accumulate aflatoxin as the culture grows but this quantity is small 33 compared to the accumulation of aflatoxin in the medium. Also, there is no difference between the amount of toxin retained by mycelia fiom shake and stationary cultures. These data indicate that there is some mechanism that eliminates aflatoxin from the filngal cells although it is not clear whether this mechanism is active or passive. Northern hybridization analysis Northern hybridization analyses were performed on total RNA extracted from each strain grown for 48 or 72 hours under shake or stationary culture conditions to determine if the genes detected using Southern analysis (nor-1 and ver-lA) were being expressed. Six probes (listed in Table 3) were used. Unfortunately, the afl-R probe only hybridized to ribosomal RNA in all of the samples (data not shown). The B-tubulin and pyrG gene probes were used in order to demonstrate that the amounts of RNA loaded in each lane was consistent (Figure 6 and Figure 7 respectively). The size of the RNA transcript detected by the B-tubulin probe for the shake cultures was approximately 1.3-kb (Figure 6a). This is consistent with the transcript size reported by Trail et a1 (1995) for cells grown on Reddy medium. In this same study in cells grown in YES medium no transcript could be detected at any time after 24 hours (up to 72 hours). Also, a 3.5-kb transcript was detected in some of the shake cultures (2112 at 48 and 72 hours, 1059 at 72 hours, AS at 72 hours, 2115 at 72 hours, and 1273 at 48 and 72 hours) that was not detected in the previous study (Trail). There did not seem to be a pattern to which strains did or did not produce this transcript. For example, some aflatoxin producers expressed this transcript early and late (1273), whereas others only produced it at one time point (1059), and still others not at all (SU-l). The suspected aflatoxin non-producers were also in this category. The unpredictable appearance of the 3.5-kb transcript may be related to 34 Figure 6. Northern hybridization of A) shake cultures, B) stationary cultures, and C) AO shake and stationary cultures using the B-tubulin probe. 35 Figure 6. C) AO 36 Figure 7. Northern hybridization of A) shake cultures, B) stationary cultures, and C) A0 shake and stationary cultures using the pyrG probe. 37 Figure 7. T. Nb ’0' >2?“ xi" «"r "o \6’ No) A!" , ,9 ,9 ,0, CV ’\ V“ \ 4.4 )A0 38 differences in the way these species regulate transcription of this particular transcript but do not reflect difl‘erences in growth of RNA in each lane. The stationary cultures differed from the shake in that they produced a primary signal at 1.7-kb (Figure 6b). The stationary cultures produced a secondary band (4.1-kb) that was also bigger than its shake culture counterpart. The absence of a pattern for the production of this secondary signal exists in these cultures too. Strangely, many of the strains that did not produce any of the secondary transcript in shake culture did produce it in stationary culture (ie. SU-l at 48 and 72 hours). If the speculation that these secondary band differences are the result of differences in regulation then it may also be true that the conditions that the cultures are grown under may greatly influence transcription. The implications of this are that stationary cultures (which more closely mimic the conditions the fungi encounter on host plants) may be more appropriate for the study of the mechanisms controlling aflatoxin production. RNA isolated from shake and stationary cultures was also analyzed with a pyrG probe (Figure 7). The size of the pyrG transcript in shake culture was measured at 1.4-kb which is consistent with the results seen by Trail. Likewise, the size of the signal produced by the stationary cultures (1.2-kb) was reasonably close to the results seen by Trail though her results did not reveal a signal after 40 hours. There may be no significant difference between the sizes because the discrepancy may be an artifact of measuring or gel handling. Once again, this main band shows that the loading of RNA from lane~to-lane was consistent. Northern analyses also were performed on RNA isolated from each of the strains grown for 48 or 72 hours in shake or stationary cultures to identify transcripts that 39 hybridized to a nor-1 probe (Figure 8a, b, and c). The shake cultures of the aflatoxin producers contained transcripts of approximately 1.0-kb whcih hybridized to nor-l. SU-l appeared to accumulate the most transcript followed by 1059 then 1273. Higher levels of transcript were detected at 72 hours as compared to 48 hours. These results are consistent with those observed on SU-l (Trail). The suspected aflatoxin non-producers did not produce detectable levels of transcript. This indicates that the nor-1 genes tentatively identified by Southern hybridization analyses are not actively transcribed in these strains. The suspected aflatoxin non-producer 2112 also shows a widely dispersed signal at approximately 0.2-kb with the nor-1 probe. This signal also occurred under these culture conditions for every probe used suggesting that an impurity in the RNA might account for this non-specific hybridization. Several independent extractions of RNA fi'om this strain in shake culture showed this “blob” during northern hybridization analyses. This combined with the fact that this “blob” did not appear in stationary culture suggested that the problem was, in fact, not caused by impure RNA. Based on the fact that this signal only occurs at the 48 hour time point suggests that this it is an RNA degradation product that is completely degraded by the 72 hour time point. This theory could be tested by conducting northern analyses at earlier time points and time points between 48 and 72 hours. The presence of this “blob” is still a mystery that, because it appears to be non-specific, may not warrant further investigation from the standpoint of aflatoxin biosynthesis. Figure 8b shows that the same pattern of hybridization occurred with the stationary cultures except that the 0.2-kb hybridization did not appear. . Northern hybridization analysis was also used to show the pattern of expression for the ver-l transcript in the seven strains for shake cultures, stationary cultures, and A0 40 samples (shake and stationary) (Figure 9a, b, and c respectively). In shake culture only the aflatoxin producers had transcripts similar in size to that reported for the ver-l (1.0-kb). Higher transcript levels were observed at the early time points (48hours). Again, SU-l produced more transcript than 1059 and 1273 (second and third respectively). Once again a “blob” appeared at approximately 0.2-kb for 2112 at the 48 hour time point. Unexpectedly, a similar signal was detected at the 72 hour time point in 2112, AS, and 1273. These “blobs” did not occur with any probes other than the ver-lA nor did they occur in stationary cultures. This combined with the fact that it is a late time point (ver- lA being a late gene in the pathway) may suggest an altered form of transcription (potentially the regulation) involving ver-lA or simply a non-stable transcript. The ver-lA hybridizations using the stationary cultures revealed a different pattern. The major signal remained at approximately 1.0-kb but the 72 hour time point appeared to have more transcript present. This shift from more accumulation at the early time point to more accumulation at the late time point may indicate that shake cultures begin to produce aflatoxins earlier than the stationary cultures. A secondary signal was also produced at roughly 4.5-kb. This secondary signal appeared to be present only when the primary band was strong. For example, SU-l produced the most intense band at 1.0-kb and had the most intense secondary band. The 1059 samples had much weaker primary signals and barely detectable (if at all) secondary bands. Even more extreme, the 1273 samples produced barely detectable primary bands and no signal was present in the area around 4.5-kb. 41 Figure 8. Northern hybridization of A) shake cultures, B) stationary cultures, and C) AO shake and stationary cultures using the nor-1 probe. 42 Figure 8. 0 A 43 Figure 9. Northern hybridization of A) shake cultures, B) stationary cultures, and C) AO shake and stationary cultures using the ver-l probe. 44 )AO 45 Discussion This study showed that Aspergillus species which are deficient in aflatoxin production under the conditions studied, do possess genes likely to be homologues of nor- 1 and ver-lA (known to be involved in the synthesis of aflatoxin). Among the species represented were the industrial fermentation species A. oryzae and A. sojae. The aflatoxin producers (SU-l, 1059, and 1273) which also have genes homologous to nor-1 and ver- lA (as determined by Southern analyses) were confirmed to produce aflatoxins. The production of AFB, (per milligram of mycelia) by these strains was greater in stationary cultures than it was in shake cultures (30 fold for SU—l). These data suggest that quantitative studies using cultures grown under shake conditions may provide an underestimate of the actual aflatoxin producting capability. Under both stationary and shake culture conditions, SU-l produced more toxin than 1059 or 1273 at the same time point. Toxin production by 1273 was greater than 1059 in stationary cultures (48 and 72 hours), whereas the opposite was true in the shake cultures. Northern analyses showed that a transcript pattern similar to previous reports (Trail et al, 1995) for aflatoxin producers was detected in the SU-l, 1059, and 1273 strains. First, SU-l cultures produced more transcripts capable of hybridizing the nor-1 and ver—lA probes than did the 1059 and 1273 cultures. These results were true for stationary and shake cultures at 48 and 72 hours. The 1059 shake cultures appeared to produce more transcript than the corresponding 1273 shake cultures. This was also true for the stationary cultures even though the 1273 cultures produced more aflatoxin under 46 these conditions. Finally, it was shown that none of the aflatoxin non-producers (SRRC 2112, 2115, A. sojae, A. oryzae) did not exhibit transcripts of equivalent size to those produced by SU- 1. In fact, no signals were detected in the stationary cultures of the aflatoxin non- producers. A “blob”, however, was detected in the 48 hour shake culture of 21 12 using all of the probes listed in Table 3. It is likely that this non-specific product is not important to aflatoxin biosynthesis based on its lack of specificity. Similar sized blobs occurred for two other shake cultures (2112 and A. sojae at 72 hours) but they only appeared when the ver-lA probe was used. This suggested that there may be some irregular expression of ver-lA occurring in these strains at the later time point. This signal does appear to be degrading which indicates a non-stable transcript. This non-stable transcript may be degraded as the result of premature termination of transcription due to a loose association of RNA polymerase with DNA, or a mutation in a ver-lA like gene. Because there was no accumulation of versicolorin A (the intermediate converted in part by ver-lA to sterigmatocystin) nor any other intermediates in the pathway when growing this strain, it is likely that this transcript is the result of abnormal transcription, RNA processing, or RNA stability. Whether this abnormality is caused by a mutation in the promoter or coding region of the ver-lA gene or in a regulator is not known. An investigation (see Appendix C) of this transcript may be helpfill in the identification of the factors involved in the secondary metabolism of aflatoxin. Much has been written on the topic of secondary metabolites. Discussions about the purposes of these compounds and their origins have been at the forefront of the speculation. This study attempted to shed light on these questions as well as to investigate 47 aflatoxin production in suspected aflatoxin non-producers. One of two possibilities seemed likely, either these strains never had the ability to produce aflatoxins or they lost that ability. By demonstrating that these strains do have both early and late aflatoxin genes it is reasonable to say that they did produce aflatoxin at one time. So what happened to aflatoxin production? To understand what happened it is imperative to ask why any strain would produce aflatoxins. It is generally thought that the production of a secondary metabolite facilitates growth and/or propagation of the species producing it (see Gene vol 115). For example, it has been speculated that antibiotic production provides strains of Streptomyces a defense mechanism against other microorganisms (Williams et al, 1989). Likewise, the production of sclerotia (wintering bodies) by species of Aspergillus provides a means of propagation for those species and possibly protection from predators - they are difficult to consume (Wicklow, 1983). Currently, the belief is that secondary metabolites were “created” as the result of gene duplications of primary metabolism pathways. This idea is based on structural similarity, amino acid identity, and organization (clustering) of genes when comparing primary and secondary pathways (Mapelstone et al, 1992). Subsequent random mutations of these genes produced various compounds that either provided a selective advantage to the organism or did not. Those organisms that did gain an advantage were better able to cope with their environment and survive. The aflatoxin biosynthetic pathway seems to fit this gene duplication theory. Based on the amino acid comparisons set out in Appendix A, the fatty acid synthase genes are closely related to their counterparts from S. cerevisiae involved in primary metabolism. So at least part of the aflatoxin pathway appears to be a mutated copy of a primary 43 metabolism pathway. It also seems that mutations occurred by chance to produce a beneficial (to the fungi) compound. Why do many species apparently possess these genes? Once again there are two likely explanations to answer this question. The first is that the production of aflatoxins is very old. So old that at one time there was only one strain that produced it. Differentiation of that “progenitor” strain (perhaps due to dispersal to various climates) resulted in many species with the same aflatoxin production abilities but distinct morphological and growth characteristics. Another possibility is that the aflatoxin pathway was foreign to many or all species of Aspergillus. In this case, vertical or horizontal gene transfer provided dispersal of the aflatoxin pathway to the several Aspergillus spp. either from an Aspergillus specie (vertical) or from an organism outside the Aspergillus genus (horizontal). Amino acid and nucleotide analyses of genes involved in aflatoxin synthesis has revealed that genes from different species share a high degree of identity. The afl-R genes from A. flavus and A. parasiticus, for example, are nearly identical showing 95% identity at the nucleotide level (Payne et al, 1993). In contrast, Appendix B shows that the pyrG (primary metabolism gene) from Aspergillus parasiticus is only 78% identical to that of A. nidulans at the amino acid level. This indicates that the pathway has not been in the individual species for very long or that genes involved in AFB, synthesis are even more highly conserved than some basic cell functions (unlikely). This lends credence to the gene transfer hypothesis which could have occurred much more recently than the divergence of a progenitor strain. One method for a horizontal gene transfer would be by a viral infection. Currently, there are few reports of viral particles found in Aspergillus spp. (Schmidt et al, 1986; Wood et al, 1974). Nevertheless, horizontal gene transfer should be a detectable 49 possibility. As has been done for Streptomyces (Vining, 1992; Bibb et al, 1989; Sherman et al 1989), secondary metabolite genes can be examined for codon usage, %GC content, and amino acid identity and then compared to other species from the genus Aspergillus and other genera. These analyses should point to the origin of the pathway whether it was completely foreign to Aspergillus spp. or if it spread from one species of Aspergillus. Why do some strains produce aflatoxin and other strains, which apparently have at least some of the aflatoxin genes, do not? Ifthe hypothesis that secondary metabolites provide a selective advantage is true then what happens if the pressure that produced the pathway is removed? Bennett and Goldblatt (1973) claimed that if an aflatoxin producing strain is grown in a laboratory culture and repeatedly passed to fresh medium that aflatoxin production is lost. This production, however, can be restored by growing the fungus on a plant. This may suggest that the selective advantage conveyed by aflatoxin production involves colonization. It is unclear how this could be, due to the fact that secondary metabolites are usually not produced during primary growth. Nevertheless, the most important information that can be taken fiom this observation (from an evolutionary standpoint) is that when the fungus is given everything it needs to grow (nutrients and conditions) in a non-competitive environment it stops making aflatoxins. This may, in fact, be what happened to some domesticated fermentation strains of A. oryzae and A. sojae. Kurtzrnan et a1 (1986) have reported that DNA reassociation studies indicate that A. oryzae and A. sojae are directly “descendant” from A. flaws and A. parasiticus respectively. Wicklow (1983) also addresses this point by noting that A. flaws and A. parasiticus strains begin to resemble A. oryzae and A. sojae during passage. (Interestingly, Wicklow also observed the loss of sclerotia production by strains that had 50 been passaged.) It is therefore, entirely reasonable that if the pressure that created the aflatoxin pathway was removed long enough, silent mutations to the aflatoxin pathway may have accumulated. This may have lead to the knock out of the pathway’s activity. To that end, it has been shown (Cotty, 1988; Diener et al, 1987) that the species most often found associated with crops (with the exception of peanuts) is A. flavus, whereas A. parasiticus is most often found in the soil (with the same exception). As stated earlier, not all strains of A. flavus produce aflatoxin, whereas all but one strain of A. parasiticus produce aflatoxin. It could be possible that crops such as cotton and corn place less pressure on the A. flavus strains that inhabit them than does the soil which A. parasiticus inhabits. This would explain why A. flavus aflatoxin non-producers are not selected against on crops. It would also explain why all A. parasiticus strains continue to produce aflatoxins. APPENDICES APPENDIX A Appendix A - Sequence analysis of the fas-lA, as-2A, and clone 6 Understanding how aflatoxins are produced was the most important aspect in determining what approach to take to eliminate them. Characterizing the conditions of growth and aflatoxin production led to the belief that removing aflatoxins from the food chain would not be accomplished by classical methods (farming techniques) due to the ubiquity of the producing strains. As a result it was necessary to identify the genetic makeup of the aflatoxin biosynthetic pathway. The first gene identified was the nor-l gene. This gene was isolated by complementing a mutant strain (ATCC 24690, nor-) with a fragment of DNA containing a wild type copy of the gene (Chang et al, 1992). The next gene to be identified was ver-1A(Skory et al, 1992). Shortly after the isolation of these genes a 35-kb fragment of genomic DNA, the cosmid NorA (Figure 10), capable of complementing both of these mutants was defined (Trail et al, 1995). Restriction analyses of the NorA cosmid yielded 11 subfragments (clones). Using parts of each of these clones as probes, northern hybridization analysis was carried out to determine the number and size of the transcripts being produced by this cosmid. In addition, these analyses were carried out at various time points to determine the timing of expression. To associate the timing of expression of each of the transcripts with production of aflatoxin, ELISA analysis was performed as described in the materials and methods (Trail et al, 1995). In a collaboration with researchers at the SRRC ARS USDA, 10 of the 11 clones from the cosmid NorA were sent to DNA Technologies (Gaithersburg, MD) to be sequenced. Analysis of this sequence has revealed the presence of a putative fatty acid 51 52 synthase (fas-lA) encoded by clones two, one, and eight (Mahanti et al, 1996). Comparisons using TFASTA, GAP, and PILEUP fi'om the Wisconsin Genetics Computer Group (W GCG) software package turned up high identity, at the amino acid level, with the Saccharomyces cerevisiae fasl gene (Schweizer et al, 1986). Identification of each of the functional domains (acetyl transferase, enoyl reductase [Mahanti et al, 1996], dehydratase, and malonyl/palmityl transferase [Mahanti et al, 1996]) found in the S. cerevisiae gene was accomplished in the fas-lA (Figures 11, 12). The fatty acid synthase in S. cerevisiae is actually composed of two subunits, the beta subunit (fasl, mentioned above) and the alpha subunit - the fas2 (Mohamed et al, 1988). The fas2 encodes the final three functional domains needed for fatty acid synthesis (acyl carrier protein, fl—ketoacyl reductase, and B-ketoacyl synthase). Again, sequence analysis of a region (clone2 and the 11th clone “4.6" [sequenced by Trail]) of the cosmid NorA showed high identity to the published fas2 at the amino acid level (Figures 13, 14, 15). The order of the domains in the S. cerevisiae genes is identical to that of the A. parasiticus genes. Also, the spacing between the domains and the active site amino acids are virtually identical between the two species. The comparisons also included an alignment with the Streptomyces antibioticus polyketide synthase which has been implicated in the synthesis of oleandomycin (Swan et al, 1994). The nucleotide sequence of the putative alpha subunit (fas-ZA) is given in Figure 16. Being that regulators typically act at promoter regions it is interesting to find that fas-lA and fas-2A appear to be transcribed divergently (Figure 10). Furthermore, based on sequence analysis, the domains from each of these genes that are closest to each other (the acetyl transferase in fas-lA and the acyl carrier protein in fas-2A) may have 53 translational start codons within 200 base pairs of each other. This leaves a rather small area for regulator binding and may suggest that the promoter regions of these genes overlap or are shared. Finally, sequence analysis of the sixth clone from the cosmid NorA has revealed a match (Figure 17) with 188 amino acids (including a heme binding site residue, cystine) of cytochrome P450 from rabbit (27% identity, 49% similarity) and rat (26% identity, 46% similarity). This gene is likely one of the many oxidases used in the production of aflatoxins (Bhatnagar et al, 1992). Keller et a1 (1995) demonstrated that the ver-lA homologue (sth) in A. nidulans requires a functional stcS to convert versicolorinA to sterigrnatocystin. stcS, located approximately 2.0-kb from sth and transcribed off the same DNA strand, is proposed to be a cytochrome P450. This situation is similar to that seen between ver-lA and the clone 6 cytochrome P450. It is very likely that as sth and ver-lA are homologues so too are stcS and the gene encoded by clone 6. Once again an interesting note bares mention. Figure 17 shows that there is a match with cytochrome P450 from rabbit (Johnson et al, 1990) and rat (Chen and Hardwick, 1993) but the comparison with the fungus Streptomyces carbophilus (W antanabe et al, 1995) does not show a significant match (17% identity, 35% similarity). This raises questions about the function of this gene. Does it play a role in aflatoxin production? If so, the presence of this gene may suggest that the biosynthetic pathway did not evolve from another pathway in the organism such as a PKS. It would support, however, the theory of gene transfer. The nucleotide sequence of the cytochrome P450 like sequence of clone 6 is presented in Figure 18. 54 Figure 10. Map of the Cosmid NorA. 55 535.0 3336 55:98:... 5.56 539—. 9.3. 80 <75» . _ _ m x 53.. 5.0.“ 5:.“ avfi.» $885 none—D 5835 65." I a: I .— ==5= I I ix I x as I H 5:... 8%.: 5.... an E. s. a. re. :3 an! __ x Hana ovfii .534 5..." 936 5...? guano—U nose—U nose—U Nose—U 0.3—0 oz 536 mos—U < az 2680 2:. 56 Figure l 1. Amino acid comparison of the suspected acetyl transferase domain of fas-lA of A. parasiticus (AP) with S. cerevisiae (SC, a FAS) and S. antibioticus (SA, a PKS). The A. parasiticus acetyl transferase domain was 27% identical and 42% similar to the same domain in S. cerevisiae over 279 amino acids, but only 16% identical and 42% similar to the acetyl transferase of S. antibioticus over the same stretch. The active site residue is marked by a down arrow. Regions of the SA were removed to optimize the alignment. Those regions are marked by “> <” and correspond to the following stretches of amino acid sequence in the published SA: 729 - 738, 811 - 829, 867 - 882, and 2333 - 2355. 1 . .4 . .3 l--l- (AC CLCL) 00¢ 2-( 11¢ -—_l '03” LLH YLK DYA i L xrxsEA “infirm” H 9.1.1 5mm exam 279 PrRfl tom HflG T6130 sflv im 'v'v P p P AP SC SA Figure 11. 58 Figure 12. Amino acid comparison of the suspected dehydratase domain from the fas-lA of A. parasiticus (AP) with S. cerevisiae (SC, a FAS) These two amino acid sequences lined up over 196 amino acids resulting in 27% identity and 43% similarity. Two stretches of the SC were removed to optimize the alignment. Those omissions are marked by “> <” and correspond to the following regions in the published SC sequence: 1456 - 1480 and 1425 ~1435. 47 I VG VG R O ipnscu cuvror KPL KPL iv GA LG YR CP p FF FF 3! YKM S S K T V v T E IHRLX VHLSN ENMX KPV L L KCDLLD DGDLLK SAR SRN VSS PNT LTR IIK A G E Aw‘ GL V V It: FA D D p LAP Y D 1 AP SC AP SC .- u «2 v2> a -< P)" 1- '>' >0 -n. z- -u.| on -2 t-‘> ar- 00- an mu m an: Zl- 00- (>- Iun. (0 ILA or: >Iu 4.: >> at! ul< — x— -n. on. o -z x -m > _J_J v30) -J> Amlu a: no (0 ml Iv-Iu _l_l GO (DI- KO (8 r—r— vb! A80 Al'- Q22 E 9 X E AP SC Figure 12. 60 Figure 13. Amino acid comparison of the suspected acyl carrier protein domain from the fas-2A of A. parasiticus (AP) with S. cerevisiaefasZ (SC, a FAS) and S. antibioticus (SA, a PKS). The A. parasiticus acyl carrier protein domain was 34% identical and 56% similar to the same domain in S. cerevisiae over 271 amino acids, but only 13% identical and 27% similar to the acyl carrier protein of S. antibioticus over the same stretch. The active site is marked by a down arrow. Two stretches of the SC were removed to optimize the alignment. Those omissions are marked by “> <” and correspond to the following regions in the published SC sequence: 336 - 344 and 359 - 370. 61 142 ASEPP 38 TP GE I AISNEPA LVRTEAA PASAVF 2 “TAEH LNAEL YA V K AM GT RL DL DY LL LL B Lu 0 TT T I L zap x0- SUD( O< POM P(( o - com om< 44 .6 a<—I uJuJ - 40¢ E‘tfi <> a :0 xi- 2 I-OO l-¥ G G W W AHLAN KYLQT RFLQS fl TV .MNE . T l PMTE Y Y 271 “i SL L L [M QSL A A M W WWN SWN S u PD 8 R T DVKKS SRKKA HSPME ATSF 2 3 9 AP E FIAG SC F FVNG SA . . . Figure13. 62 Figure 14. Amino acid comparison of the suspected B-ketoacyl reductase domain from the fas-2A of A. parasiticus (AP) with S. cerevisiae (SC, a F AS) and S. antibioticus (SA, a PKS). The A. parasiticus B-ketoacyl reductase domain was 42% identical and 62% similar to the same domain in S. cerevisiae over 479 amino acids, but only 14% identical and 34% similar to the B-ketoacyl reductase of S. antibioticus over the same stretch. The active site is marked by a down arrow. One segment from the SC and SA was removed to optimize the alignment. Those omissions are marked by “> <” and correspond to the following regions in the published sequences: SC, 946 - 1012 and SA, 915 - 2937. 48 GLB.. HL KK GASF TIPF FRE PR [ii IAYIQ Tvssr p K “mans A A SL Y V E TI 1" TKD DKD I L ( g Ql-ILO pax< "EOE 0:03] mm < 000: 02‘ 1'2) was 0x4 -> ODD HEM wwa <49 <l- 0 >-E m I->-0 J .1 -— UL) - xxB & x on! 2.1 . EB; > »-< i~~< (w- >u= x I] ra-a -- F F MYD NH IYA VRB LE 88 A osrP SF PF Econ CGA chm - Ono—I COB a—a mh> -0 44w . >->-< X< 00: fig. 3;; I 5i” (o- 20> DO - _J_i( .Iu. - 49> (a- 2- am -x h»- mm- mm- -- P>- ox- >3- OD . EE- ”<- a .n, . v-h $85 Figure 14. XRAR . .BSF .AD K N 3 MG m mGXARVY EWGSARTK ”Elsvo AAA WSGNSEAG VVTGFAEVG HAAGVPEST LEE EPM KE .TA K W I C C G V -- 3.1 0» Figure 14. 65 Figure 15. Amino acid comparison of the suspected B-ketoacyl synthase domain from the fas-ZA of A. parasiticus (AP) with S. cerevisiae (SC, a F AS) and S. antibioticus (SA, a PKS). The A. parasiticus B-ketoacyl synthase domain was 41% identical and 61% similar to the same domain in S. cerevisiae over 631 amino acids, but only 19% identical and 37% similar to the B-ketoacyl synthase of S. antibioticus over the same stretch. The active site is marked by a down arrow. One segment from the SC and several from the SA were removed to optimize the alignment. Those omissions are marked by “> <” and correspond to the following regions in the published sequences: SA, 52 - 60, 101 - 108, 318 - 334, 341 - 434; SC, 1435 - 1520; SA, 461- 468,1709 -1715, 1807 -1812, 1843 -1850, 1954 - 1960, 1996 - 2007, 2049 - 2067. (m um mm mm m mfi >< SF KV EA 1 AP PIFBD RF..D DPDflD 0F AL LY flPR Pm .GR i Y L ATL WDL ch one VK. VKL LPE ovc c v eiA P P EIP r N on L E L A D G RLREE G BHQ REP 0 U) SA «EEO QIIK RHI ..V .AL FBFDi EMYKY YPL DPY Rn. m K FY Fl FE TIFEVAEA VLV VVEA LLETSWEA L Y LF L 28$ EABGC EA GA 6 AAEGV AV N6 06 E A Pmne PATT L R K EH E SR E E IL VNVE SNTL . .TT T T V A R K I M a: F F u FE L. cent/)1- 9(0) ..-.. U( 0.0 n. m fin» «m5 <88 <85 8% Figure 15. RPHES HRVLAEV Figure 15. 68 Figure 16. Nucleotide sequence of the fas-2A of A. parasiticus. 69 .2 95m; OOBUOBUO¢UOH¢U¢0000¢98¢¢U0800028004008UCUOUOU‘BBOUZ‘U‘OBH‘OHUGOOUBUUdaIduoavfiuoufioaflzOUUOH¢4¢HOO¢UUU ”MOW0dOUUGOUOOOO‘Z‘UUUGOB‘U‘UOQHBHBO‘OBO‘OHUOUOBOHUOU‘HBBOOOOQBU900900cadoUB‘OUdUBB‘OOBflUUO‘UGBHWMMM “WWWG‘OOOOU‘dUU‘U000000880900afituddufioaOOO‘OOH008000OU¢UGO0809000¢0¢UH¢0¢G¢UO<¢UOO¢BUOUOZHUGOCOOMWWU MMWNOBOO£UHBOOUB¢¢GG¢OU¢OB00¢094¢¢0004‘0006OEOBBUUOOOOUGBBO¢OO¢OOUOO¢ZOOUOOBBO¢HUdflU‘dflUO‘BUOU‘UMWMM WMMWOOBOU¢¢OBOUOU‘BO‘B‘UOwBBOO¢OBOOO¢¢GOBHUOOHOUzUOOCUB‘OOOOOQCB900002002008GB¢O¢UOUHUOOUU£UU¢UHMMMW WMMWBUOOBBBH6000280080090000U9‘0000000029¢<4¢¢0¢009<000¢40ZQBQO¢B¢0¢OOUBU084000000BUOOOBUFO‘B‘UOWWMM ”mmwocuadu2090¢O¢00000¢OOB¢OBdOB‘UO‘O‘UZUBOO‘UUUUUHU880009GOOHOO¢BU¢OBBOOUUB‘OCUO‘OQOUGCOUB‘UOBUWMWM ”WWNUwOBUGOUB‘B‘O‘U‘OUOOOZOOUHOUU¢OUUOO¢UOHUG‘UO“UO¢U¢DOOU¢UQ¢¢UOH0008(80880BH‘UUGB‘UflddofldoaowwwmM “WM“BOOO‘HBUUOBUOOUOB‘doUOUdOBHBBOHO‘UUUUH‘O‘00000600UG‘H‘UUOBQ‘OOO‘OO‘O‘U09898080000040‘4098H‘Oflwmw “MMB690911680800000000000090UUB‘UBBHUBOH“UBUB¢OO80684008004839000008000BBBO‘GBHUU‘H‘OGOH¢¢U¢¢OH‘WMN “MMBHH‘GOH‘OUB‘UBO‘080808698UBOO‘UOOUUO‘UU‘UOOUHBH$800000000009800Houafldouuakooauduefia0¢OHOUHQOHUWMM “WWOOHBUOH‘UOOO‘Oa49000U(DU000‘0OUO0‘10BdUtUfldOdUU‘dOfidfiB‘OBU‘UO‘QOOOZGOBOH‘ddouH00¢OB¢OH¢B¢OOUB¢WMM MM”00¢U¢B¢¢U¢B090BO‘ddfidfiudficflflafiufifldu940880400¢OUO¢OOBH¢O¢CUQBUO98100B‘douaflflaadduUG‘OG‘BO‘OBOBdwwm WWMdfifitUOZOB‘OBOOOHH¢¢HB000‘090009000‘009904000OBOBOOU¢¢BOBOBOOO000ZO¢OH¢4800¢BG&OUGOBdfldfiddO‘UO‘WMW WMMUH‘G‘OOOOOOOOOOOOOOO‘BO‘U¢U¢0¢H¢BO¢¢OUO08000090‘0‘4440800¢ZQ¢008(‘0‘08<¢UO¢¢UOB¢OO¢4¢OZ008ZUOBWWM WWW‘ddtufittuoaflataaaddoad00002909¢49¢¢0000040000002Edda0‘880(808‘80‘0‘89‘408800109¢O¢09¢4808¢¢00¢WMM MMM0440¢d4¢000900¢00110006¢00080¢980(8044‘904898UGOQ‘OOUOHOUOOBOB‘ZBHBO0090900409800008‘000‘ddoBBNMW OOH . H 7O .2 05mm HOOH‘UHOGF‘OBUBUH¢£U¢¢O¢94UUOUUBB09889UG‘BUOG‘UOBHUUBUOOO‘UUUGUOHH‘OUBUOUUOOOBO‘BOUG‘dUU‘UflHUUOflB‘UU WWWMOCOOUOOH‘dfitu900880BUB‘dOUOOOOUdO‘OUUOBOOUG‘UOBHBOOddOOHBUH‘GOOOUGBB‘OUdOflOO‘UUflGOOOdHBO‘UOU“WWW “WWMUDDB‘OOOO‘HBOUOOOUUZ‘CBO0UflooUHBB‘BUHOBUUOHUOOZUBOCUUOBU¢¢UBDUOOO¢¢8404GOBBG¢¢000¢UUO¢UH€UBBWMMM MWMMOGGBUC‘UBBOOB‘UBB‘OOCHUU‘OUUG‘OOU‘OUEda0 “PX ' 3.1! 01th I: - 334 no< a.” flfll 20H: 00-0 I-tn— uuz tzlz< a wmn 33— Oh! I — tn>- >-iu >0< DOD .ri-i- «:0 “hi mam flan 4!!! °>DI I, lll~~ w - - - 2 in In in 2 2g 3 2 2 2 gigs a 5; 5; E: :2 c E a t 553 552 5:: 3 “En. l"Eta a.‘ZIL .35 5 dc: d”: d“: 34.5.; " ('an ('dae <4... I El protein (ACP)-synthase and (B) aeyltransferase domains of gene-l and polypeptides in the GenBank and EMBL databases. Amino acrds Identical in at least two of the presented sequences are shown as white letters on black backgrounds. When two different pairs of Identical amino acids occur at the same residue. the pairs which do not match the A. parasiticus (A9.) I I V I I sequence are shown In Italit: Arrows in panels A and 3 Indicate active-site cysteine: and sennes. respectively. See text for analysis of comparisons. IAInL . .— r 1 "‘1'" r FIG. 7. 2672 TRAIL ET AL. wA PKS (32%) and rat fatty acid synthase (FAS) (20% [SI]) enzymes and a distinct region in the gene-l sequence (Fig. 7B). The identity of gene-I in these two functional domains was higher with PKS than with FAS. suggesting that gene-I en- codes a PKS involved in AFB] synthesis. However. these data do not rule out the possibility that gene-l encodes an FAS. Limited nucleotide sequence analysis of m-m8 (37) identified a l80-amino-acid region with a high degree of sequence iden- tity (48%) to a region of undefined function in FASI genes encoding the beta subunit of FAS in the yeasts Sacclmromyces cerevisiae and Yarowr'n lipolyrr'ca (3|). Townsend et al. (53) proposed that six-carbon hexanoate (two kcto groups com- pletely reduced to hydrocarbon) may serve as the starter unit for AFBI synthesis and that hexanoate is extended by a PKS without further ketoreduction to form a decakctide. NA. This scheme would include at least one multifunctional enzyme. the FAS. with the necessary activities to reduce keto groups to hydrocarbons in order to synthesize the hexanoate starter. Another set of activities, the PKS. without kcto reduction ca- pability (27). would then extend hexanoatc to generate the decakctidc. NA. Our limited data are consistent with this scheme. uvnr8. which has a high degree of identity to yeast FAS. could fill the hypothetical FAS role to produce hexano- ate. which is extended by the product of gene-l (pks/i). a putative PKS. In support of this theory. Chang et al. (I4) have independently disrupted and sequenced more extensive re- gions of gene-l. which they have called pksA. Several func- tional domains associated with polymerization of acetate (B- ketoacyI-synthase. acyltransferase. and acyl carrier protein) were identified. but III‘ analysis strikingly failed to find evi- dence for a kcto-reductase. dehydratase. or enoyl reductase involved in reduction of kcto (carbonyl) groups to hydrocar- hon. A second approach. gene disruption. clearly demonstrated that gene-I activity occurs prior to the ten] gene in the patlv way. Further evidence for gene-I function is provided by the studies on sclerotium production in gene-l-disrupted transfor- mants. The absence of production of sclerotia by strains that accumulate pathway intermediates between NA and VA sug- gests that gene-l is involved at a step prior to nor-I activity. In a separate study it was shown that strains disrupted at III'HIH, like strains disrupted in gene-l, produce sclerotia at levels higher than those produced by SUI (36). Since it was demon- strated that uvm8 activity occurs before nor-l in the pathway. this would argue that gene-l. like ui'm8. is involved in some stage of polyketide backbone synthesis. In previous research. an association between aflatoxin bio- synthesis and sclerotium development has been observed (6, l8. I9). Using a molecular genetics approach. Skory et al. (48) observed that complementation of the tier] mutation in strain A. parasiticus CSIIl. which accumulates VA and normally does not produce sclerotia on potato dextrose agar. restored wild- type levels of sclerotium production. The data presented here for the AVF- and VA-accumulating strains suggest that accu- mulation of pathway intermediates inhibits sclerotium devel- opment. Strains which accumulate early pathway intermediates (NA) or no pathway intermediates (gene-I and iri'm8 dis- ruptants) generate wild-type levels of sclerotia (or more). sug- gesting that elimination of accumulation of intermediates in the middle of the pathway allows sclerotial development to occur. When no intermediates accumulate. sclerotial develop- ment is apparently enhanced. Together. these observations support the hypothesis that the biosynthetic pathway for afla- toxin production strongly affects the development of sclerotia. Since secondary metabolism has long been considered a form of metabolic differentiation (7). it is not surprising that it may 86 Am. Eavraoa. MicrtoaioL. also be linked to morphological differentiation. Just how this link is structured remains to be elucidated. ACKNOWLEDGMENTS This work was supported by a cooperative research agreement with the USDA and grant CA52003-0IAI from the National Cancer Insti- IUIC. REFERENCES I. Anderson. J. A.. and l. I). 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Environ. Microbiol. 60:25tiI—25o7. Maynrga. M. E.. and W. E. 'l‘ltnherlalte. I992. 'Ihe developmentally regu~ Iatcd Aqrrgillus nirlulrrns n-A gene enemies a polypeptide homologous to polyketide and fatty acid syntheses. Mol. Gen. Genet. Ll5:2ll5~2l2. Montenegro. E.. R. Fierrn. I-‘. J. I-‘ernandea. S. Gutierrez. and J. I". Martin. I992. Resolution of chromosomes III and VI of As/rrgr’llm nirlularrr by pulsed~field gel electrophoresis shim that the penicillin biosynthetic path- 87 AFLATOXIN GENE CLUSTER IN A. PARASITICUS 2673 43. 45. 47. 49. St . 5 . 52. 53. S4. 5 'J! 55a. 57. 58. way genes pchB. prhC'. and pfllDE are clustered on chromosome VI (3.0 megabases). J. Bacteriol. I74z7tl63-7067. Oakley. B. R. J. E. Rlnehart. B. 1. Mitchell. C. E. Oakley. C. Carmina. G. L Gray. and G. S. May. I987. Cloning. mapping. and molecular anal- ysis of the pyrG (orotidine-5'~phosphate decarboxylase) gene of Aspergillus nidulans. Gene 6|:385—399. . Papa. K. E. I984. Genetics of Aspergillm floors: linkage of aflatoxin mutants. Can. J. Microbiol. MW”. Payne. G. A.. G. J. Nystront. I). Bhatnagar. T. E. Cleveland. and C. P. Woloshnk. I993. Cloning of the all-2 gene involved in aflatoxin biosynthesis from Aspergr'lfus flours. Appl. Environ. Microbiol. 59zlSlv-I62. . Pestka. J. J. I988. Enhanced surveillance of foodborne mycotoxins by im- munochcmical assay. J. Assoc. Off. Anal. Chem. 7|:ltl75—lfllil. Reddy. T. V.. L. Vlswanathan. and T. A. Venkitasuhramanlan. l97l. Iligh aflatoxin production on a chemically defined medium. Appl. Microbiol. 22: 393-390. . Skory. C. I).. P.-K. Chang. J. Cary. and J. E. Lina. I992. Isolation and characterization of a gene from Aspergrllm parasiticus associated with the conversion of versicolorin A to sterigrriatoeystin in aflatoxin biosynthesis. Appl. Environ. Microbiol. 583527-3537. Skory. C. I).. P.-K. Chang. and J. E. Lina. I993. Regulated expression of the nor-I and ver-I genes associated with aflatoxin biosynthesis. Appl. Environ. Microbiol. 59: Ifi42- I646. Skory. C. I).. J. S. IInrng. J. J. Pestka. and J. E. Lina. I990. Transformation of Armrgiflm parasiticus with a homologous gene (pyrG) involved in pyrim- idine biosynthesis. Appl. Environ. Microbiol. 56:3.“5—332". Smith. S.. J. K. Naggert. B. Williams-AMI. and C. M. Amy. I992. Inlron-exon organization of the gene for the multifunctional animal fatty acid synthase. Proc. Natl. Acad. Sci. USA ”zl ItIS—I ltltl. Swan. I). G.. A. M. Rodflnfll. C. Vilches. C. Mendel. and J. A. Salas. I994. Characterization of a .Vrrrmmnv-rs urrrilmmrm gene encoding a type I polyketide synthase which has an unusual coding sequence. Mol. Gen. (ienet. 20:358—362. Townsend. C. A.. S. M. McGuire. S. W. Ilmbst. T. L. Grayhlll. K. Pal. and C. E. Barry III. l99l. Examination of Ictrahydro< and dihydrohisfuran for- mation in aflatoxin biosynthesis: from whole cells to purified enzymes. p. I4l—I54. In R. J. Petroski and S. P. McCormick (ed). Seeoodary-metalmlrte biosynthesis and metabolism. Plenum Press. Inc. New York. Trail. I-'.. P.-K. Chang. J. Cary. and J. E. Lina. I994. Structural and func— tional analysis of the nor-I gene involved in the biosynthesis of aflatoxins by Arpwgr’llm ,mmrr'mus. Appl. Environ. Microbiol. “Minx—4085. . Wulnshak. C. I‘.. K. It. Pooh. J. I-‘. Brewer. I). Bhatnagar. ’I‘. E. Cleveland. and (i. A. Payne. I994. Molecular characterization of u/IR. a regulatory locus for aflatoxin biosynthesis. Appl. Environ. Microbiol. 60:24flIl-24I4. Wu. T.-S.. and J. E. LinL Unpublished data. . Yahe. K.. Y. Matsuyama. Y. Andn. II. Nakajima. and T. IIarnasakl. I993. Stereochemistry during aflatoxin biosynthesis: conversion of norsolorinic acid to avcrulin Appl. Environ. Microliiol. 59:24llti-2492. Yu. J.. J. W. Cary. I). Bhatnagar. T. E. Cleveland. N. P. Keller. and l". S. Chu. I993. Cloning and characterization of a cI)NA from Ava-mill!" mnrsr‘lirrrt encoding an U-mcthyltransferase involved in aflatoxin biosynthesis. Appl. I:nviron. Microbiol. 59:3564457I. Yu. J.. I‘.-K. Chang. J. W. Cary. M. Wright. I). Ilhatnagar. T. E. Cleveland. (I. A. Payne. and J. E. Lina. I995. Comparative mapping of aflatoxin pathway gene clusters in Arperglllm mmsilrcm and Arpcrgrflus flaws. Appl. Eat/iron. Microbiol. 6|:23fiS-237I. 88 In the following manuscript I was primarily responsible for the sequence analysis and figures generated from that work. I also helped with the photography of the thin layer chromatography plates. 89 Arrinzo AND ENVIRONMENTAL Micaorrrorrxsv. Jan. I996, p. l9I—I95 Vol. 62. No. I (Km-ZZWISMIIHO Copyright 0 I996. American Society for Microbiology Structure and Function of fas-IA, a Gene Encoding a Putative Fatty Acid Synthetase Directly Involved in Afiatoxin Biosynthesis in Aspergillus parasiticus N. MAHANTI."( D. BilATNAGAR.2 J. W. CARY} J. JOUBRAN.'T ANI) J. E. LINZ" Department of Food Science and I laman Nutrition. Michigan State University. [first Lansing. Michigan 48824.’ uml Southern Regional Rt'M'llR‘ll ('r'nu'r. Agricultural Rowan-Ir Sr‘n'lr‘t‘. US. Dr'pamnr'nt of Agnr'ullurc. New Orleans. Louisiana 70179-06872 Received It) July lWS/Accepted l8 October I‘NS A novel gene. [as-IA. directly involved in aflatoxin BI (AF BI) biosynthesis. was cloned by genetic comple- mentation of an Aspergr'llm parasiticus mutant strain. UVMB. blocked at two unique sites in the AFBI biosynthetic pathway. Metabolite conversion studies localized the two genetic blocks to early steps in the AFBI pathway (nor-l and [as-IA) and confirmed that fas- IA is blocked prior to nor-I. Transformation of UVM8 with cosmids NorA and NM!) restored function in nor-l and [as-IA. resulting in synthesis of AFBI. An 8-Iib Sacl subclone of cosmid NorA complemented [as-IA only. resulting in accumulation of norsolorinic acid. Gene disruption of the [as-IA locus blocked norsolorinic acid accumulation in A. parasiticus 862 (nor-l). which normally accumulates this intermediate. These data confirmed that [as-IA is directly involved in AFBI synthesis. The predicted amino acid sequence of firs-IA showed a high level of identity with extensive regions in the enoyl reductase and malonyI/palmityl transferase functional domains in the beta subunit of yeast fatty acid synthetase. Together. these data suggest that firs-IA encodes a novel fatty acid synthetase which synthe- sizes part of the polyketide backbone of AFBI. Additional data support an interaction between AFBI synthesis and sclerotium development. Aflatoxins are polyketide-derived secondary metabolites that are produced by strains of the imperfect fungi As/x'rgrllus pamsiticas and Aspergillus flavus. Allatoxins are highly toxic. mutagenic. and carcinogenic in a variety of animal species and are suspected carcinogens in humans (l I). Peanuts. treenuts. corn. cottonseed. and other important crops are occasionally contaminated with aflatoxin as a result of infection by toxigenic aspergilli. An understanding of the aflatoxin biosynthetic path- way may result in the identification of strategies to inhibit aflatoxin contamination of plant-derived products at the pre- harvest level. Aflatoxin biosynthesis is preposed to begin with the conden- sation oi acetyl coenzyme A and malonyl eoenzyme A via a polyketide synthetase (PKS) to form the decaketide noran- throne (4. l0). Alternatively. a six-carbon fatty acid. hexanoate. is lirst synthesized by a fatty acid synthetase (FAS) and then extended by a PKS to generate norantlirone (22). Norantlirone is oxidized to norsolorinic acid (NA). which is converted to aflatoxin Bl (AFB!) through a series of pathway intermedi- ates. including averantin (AVN). averufanin. averulin. versico- nal hemiacetal acetate. versiconal. versicolorin B. versicolorin A (VA). demethylsterigmatocystin. sterigmatocystin (ST). 0- methylsterigmatocystin (OMST). and AFBI (4. In). Several genes encoding enzyme activities or regulatory pro- teins involved in AFBI biosynthesis in A. [mrasr'ricus and A. flavrrs and ST biosynthesis in As,mgr'llus nirlulmrs have been cloned (6. 24). These genes are clustered in a (iS-kh region on one chromosome in A. parasiticus and A. flarus (25. 26). Tran- script mapping analysis identified three other genes in the cluster encoding large transcripts (7.5. 7.0. and 6.5 kb) which 'Corresponding author. Phone: (5l7)-353-9(i24. Fax: (SID-353- 8963. t Present address: DNA Unit. Michigan State Police. E. Lansing. MI 48823. l9l appear to be involved in AFBI biosynthesis (25). Gene disrup- tion and nucleotide sequence analyses of pksA (7.0-kb tran- script) suggested that it encodes a PKS involved in synthesis of the AFBI polyketide backbone (7. 25). This study focuses on [as-IA. which encodes the 7.5-kb tran- script. Analysis of [as- IA mutants combined with nucleotide sequence analysis strongly suggests that this gene encodes one subunit of a novel FAS directly involved in synthesis of the AFBI polyketide backbone. MATERIALS AND METHODS Strains. plasmids. and culture conditions. Plasmid DNA was propagated in lsrltrmliui mlr ltllin (l2) and purified by an alkaline lysis procedure (I8). l'lasrrirrl pit/.3 K (see l’ig I) urritains a 3.3 kb liroill subclone oi cosmid NorA inserted into the 1le site of plasmid pllliiesciiptllSK( --). l’lasniid pAi'SaX (irg It contains .rrr N kb Sm l subclone oi cosmid NorA inserted into the Sari site ol pllliicsctiplllSl“ H) .-t [irrnrsrurm SUI (A'l‘('(' $1075. NRRL 5862) served as the allatosin~pro- doting \srltl-tipe strain .l printout m lilo! (rrr'al) nor-l hrl [8]). derived from .l pmmrrrr m A l(‘(. I-lo‘ll (lb). was used lor isolation ol mutants created by UV inutagenesis and for gene disruption experiments. lib! accumulates NA and retains the ability to produce low levels of AFlll (live- to eighltold less than 5U l l Methods for the maintenance of iringal strains and preparation of conidial stocks and descriptions of the liquid and solid growth media used [or production (VLS) and analssis (crieonut agar medium ICAMl [2]) of allatosins have been reported prexiouslv (35) [TV mutagenesis. (‘onidia in sterile water (llf‘ per ml) were exposed to up to I" sequential doses ol UV light at ltIUIII pJ per the: (UV Stratalinker'. Stratagene). Mutants lacking NA synthesis were obtained after irradiation with sesen doses (UVM7) or eight doses (UVMtl). Metabolite cons ersion studies. Metabolite conversion studies with whole cells were conducted as described by libalnagar et al. (5) and Adye and Matelcs ( l ). One gram (wet weight) ol washed mycelia from UVMT or UVMtl was incubated lot I: h with constant shaking ( lit) rpm) at 28°C in the presence of acetate ( Lilli pg). NA (It) pg). AVN (to pg). VA (to .13). ST (5 pg). or OMST (5 pg). Atlatosins were analvred by thin-layer chromatography (TLC) and quantitatcd by densitomcrrs (Shimadzu dual-wavelength TLC scanner model CSOIIIM) as described by Bhatnagar et al. (5). I92 MAHANTI ET AL. ear-I L's-M urn Comp 1: — a a -' “NA A J. J’A'l‘A‘A‘ a a A O f. 5 - .2.” eon-tam . .1 f . .23.. m2. 0 8 l8 ‘ 9‘” o ’f . I“III parrots - XX CosmidVer2 ...... _1La_i_ ...... ‘ Vet} i.— ...... ' XX VON ...... .—1LI—L— ...... ‘ _ -o 4% ml. FIG. I. DNA fragments used [or complementation oi UVMll. Seven DNA lragments. reprexnted by solid lines. were used in compiementation experi- ments. Only insert DNA is shown. Cornplementation (Comp) oi UVMli is shown for each fragment. Unlabeled restriction endonuclease sites are EcoRI. Other sites are Sari (s) and Xhul (x). The thick arrows represent the size and orientation ol‘ transcripts [rum nor-I. Her-M. ver-lll. and [us-M. The numbers 2. I. and Ii on the maps for eosmids NorA and Norli and subclones pAPXbZli and MM are EcoRI subclones used in nucleotide sequence analysis. The small an'onson the NorA map show the approximate length and direction of sequenc» ing to generate the data shown in Fig 6. The dotted lines on the maps oi «mitts Ver2. J. and 4 represent unmapped regions. Genetic couple's-tuba: traoslor-atiaa at fungal protoplasts and analysis i W clones. Protoplasts were transformed by a polyethylene glycol procedure (2i). Plasmid pSUiZ contains the nitrate reductase gene trial) (I3). teted as a selectable marker tor uitranslormation. The cosmids used in comple- mentation experiments (Fig. I) were isolated in a previiius study (7.5): cosmid NorA Contains nor-I and err-IA (I7); uismid Norli contains mar-I on a Zl-kb DNA lragment that overlaps with NorA; and aismids Ver2. VerJ. and Ver4 contain all or part oi a I2-lib duplication of the wr~lA aflR region on cosmid NorA. well}. a nearly identical copy oi oer-M. is «attained in this duplicated region (I7). Cosmids or subclones were added in 2- to Ill-told molar excess over 36W. mil) ' translurmants. selected by growth on C lapel: Dos (CZ) medium. were translerred to CAM to screen [or allatosin and NA accumulation. Amines of all-tul- pnduction in translornaats by TLC and ELISA. Alla- tmin and NA produced by the recipient strain 862 and [co-M disruptants were quantitated by TLC and enzyme-linked immunoaorbent away (ELISA) by the method ol' Trail et al. (2.1)eseept that cells were cultured lot 65 h instead oi 72 h. Genomic DNA labile. and Southern analysis. Genomic DNA was prepared by a published modilieation (l4) of a phenol-chlorolorm protocol developed for mammalian DNA (3). Restriction endonucleases were purchased from New England Biolabs or Boehringer Mannheim Biochemicals “:P-labeled DNA probes were generated with the Random Primed DNA Labelling Kit lrom Boehringer Mannheim Biochemicals. Southern hybridization analyses were mn- dtcted by standard procedures (3). Sclerotia- production. Approximately IO‘ conidia were center inoculated onto petri plates containing 20 ml of CAM. Plates were incubated in the dark at 28'C [or 7 days Sclerotia were harvested and uiunted by a modification (l9) oi the method ol Cotty (9). Scierotial diameters were measured with a Java video analysis system (Jandal Corp.. Cone Madera. Calif.) TABLE 1. Conversion of metabolites to AFB! by whole cells of two mutant strains ofA. parasiticus Mean AFBI produced" (pg/mg ol Metabolite m mycelium [wet mi) ("4) uvm uvm None ND ND Acetate 1.000 ND ND NA 10 0.39 1' 0.“ 0.23 3'. 0.“ AVN to 21 z 0.6 1.6 2 0.4 VA l0 3.4 t 0.9 4.] z 0.6 ST 5 3.1 I 0.2 2.8 I 0.4 OMST s 4.6 : l.0 5.1 2 0.8 ' Mean of m experiments with two replicates each ND. none detected. 90 Am. ENVIRON. MICROBIOL 1234807. FIG. 2. TLC analysis of cell extracts from UVMll complementation experi- ments. Lanes: l. UVMti; 2. UVM7; 3. 362; 4. UVMll transom-d with pAPXhZIl; 5 and 6. two UVMll isolates traml’ormed with pAPSall; 7. NA stan- dard (arrow labeled Nor): ll. AFBI standard (arrow labeled AFBI). The band immediately below AFB! in lanes 2. 3. 4. 5. and n is AFGl. Nucleotide sequence analyses. Nucleotide sequence analyses were conducted by DNA Technologies. Inc.. Gaitbersburg. M.. on three ctr-amid NorA subclones containing [as-M (clutter: 2. I. and ll [Fig II) (25). Nucleotide sequena data were analyzed with the Wisconsin Genetic Computer Grmp (GCG) soltware package. The locations of introns and open reading Irames were predicted by using GCO programs Franks TestCode. and Codonl’reierenee. Comparisons of the predicted amino acid sequence atlas-IA with sequences in the EMBL and Genilank databases were eondixted with TFastA and Gap. A Noelutidesqaeaeeaeeusiaaaambu.1‘heaceuaim numherlorlas-IA is Will}. RESULTS Isolation of UV mutants UVM7 and UVMB. UVM7 and UVMli. derived from A. pumsilicus 862, no longer accumu- lated NA (red-orange pigment) or AFBI (blue fluorescence) on CAM. UVM7 produced nonpigmented mycelia. and UVM8 produced a bright yellow mycelial pigment that was secreted into the growth medium. Loss of AFB! and NA gn- thesis was confirmed by TLC analysis (data not shown). Inabil- ity to grow on CZ medium indicated that these mutants re- tained at nonfunctional m’aD allele. Metabolite conversion studies. UVM7 and UVM8 con- verted VA. AVN. ST . and OMST to AFBI but could not convert acetate to NA or AFB! (Table l), suggesting that they were blocked prior to nor-l (the product of nor-l converts NA int-a Ecol! can M Eeall\ \(M M soil “(will a A A ‘ JD 4 a : : : : Ema 1.4-ts 1.4-b on 2.74. FIG. 3. Gene disruption atlas-M. Recombination between the 2.8-kb insert inpRZZ.8(solidarea)andthehomologousregioninles-Muolidboniathe genome generates Hindlll restriction Iragmentsol the sizes indicated on the map labeled disrupted genome (the probe is the 2.8-kb insen). Nondisrupted strains have Hindlll fragments of the sizes indicated on the map labeled genome. “l Vot_6Z I996 FIG. 4. TLC analys'n of cell extracts from Disl. -Z. and -J. lanes: I. NA standard:1 extract from 862: J. 4. and 5. extracts from Disl. -Z. and -J. respec- Iivciy. to AVN). The mutants converted five— to eightfold more AVN to AFBI than NA. suggesting that they retained the IIor-I genotype and were therefore blocked at two sites in the AFBI pathway. nor- -l and far-IA. Comp lementatiott of UVM8. UVM8 was cotransformed with plasmid pSUIZ plus one of five cosmids or cosmid subclones (Fig. I). Cosmids NorA and NorB complemented both path-. way mutations in approximately I% of the IriuD‘ transfor- mants in two separate experiments. resulting in synthesis of AFB]. Cosmids Ver2. Vcr3. and Ver4 and plasmids pAPXblS. containing a I5-kb subckinc of cosmid 698 (It). and pSlJlZ (control) failed to complement UVM8. Cloning of far-IA. A ZX-kb Xbul subclone of cosmid NorB (pAPXb28 [Fig ll). which carried the Zlvkb overlap between hese clones. complemented fax-IA and nor-I or fax-IA akine in strain UVMil. resulting in transformants which produced AFBI (6 of I60 nI‘aD‘ transfonnants) or NA plus small quan- tities of AFB! (five- to eightfold less than SUI) (l of I60 niaD‘ transformants). respectively. Comparison of the restric- tion endonuclease maps of cosmids NorA and NorB and plas- mids pAPsztl and pAPXblS localized [as-IA to three con- tiguous EcoRI subclones of cosmid NorA (clones 2. l. and 8 [Fig. H) An 84th Sad subclone of cosmid NorA (pAPSaX) containing clones l and 8 and part of clone 2 was used with plasmid pSUiZ to cotransform strain UVMH. Two of 30 niaD‘ transformants accumulated NA on CAM. suggesting that pAPSa8 complemented [as-IA (and not Imr-I) in UVMII. Con— trol transformants (pSUlZ only) did not produce NA on CAM. TLC analysis of the recipient strain and transformed isolates (Fig. 2) determined that UVM8 failed to produce detectable AFBI or NA. whereas UVMII transformed with pAPXbZil produced AFBI. UVMII transformed with pAPSa8 produced NA and AFBl at levels similar to those of the nor-I mutant A. parasiliais 24690. UVM7 produced low levels of AFBI and aflatoxin GI (AFGI). suggesting that the mutations in UVM7 and UVMS are not allelic. Gene disruption of [as-IA. Transcript mapping analysis pre— viously localized a 7.5—kb transcript to the [as-IA locus (25) (Fig. I). A 2.8«kb EcoRI cosmid NorA subclone (pR22.8 [Fig 3]) from the middle of the 7.5—kb coding region was used to disrupt fax-IA in strain 362. an NA-accumulating strain. Ap— proximately 4% of the m'aD‘ transformants failed to produce detectable NA or AFBI when grown on CAM. suggesting that they were [03- -IA disruptants. All pSL82 transformants (con- trol) produced NA on CAM. No transformants were obtained in the absence of plasmid DNA. Three putative [05- IA disruptant clones. Disl. Dis2, and Dis3, were subjected to TLC (Fig. 4) and direct competitive 91 A. PARASITICUS AFLATOXIN BIOSYNTHBIS GENE fax-M I93 l1l3¢5 ma» - c 0‘. 1.15 cog..- “.II-16 5 ISIIIIIMM .hyiiridization analys'emf Di\l. -2. and -J. Genomic DNM 2.8-kb Ele insert from pllflll a a puil'xl'. Arrowheads show the sizes of sin. \tmma ers: I 2. and J genomic DNA isolated from Disl. -1.:nd -J.ri. “ivelir. 4 and S. lemme DNA from IIiiIIJ'. NA-accuinttlating transformants (nondisntpted strains). ELISA analyses. Disl. —2. and -3 did not produce detectable NA or AFBI. whereas B62 transformed with pSUQ (control) accumulated NA and low levels of AFBI. Direct competitive ELISA confirmed that 862 transformed with pSUlZ (control) produced lull-fold more AFBI (3 to 5 pg/ml) than Disl Dis2. and Dis’l (0.002 to (LOIS nglml; near the limit of detection). Strain SUI (wild type) produced approximately 6. (Ill-fold more AFBI (ltltl pig/ml) than Dist. -2. and -3 and approxio mately 2Il-fold more AFBI than strain [361 The disruption of [us-IA in Disl. -2. and -3 was confirmed by Southern hybridization analysis with the "zP-labeled 2.8-kb EcoRI fragment of pRZZ.8 as a probe (Fig. 5). Genomic DNA isolated from Disl and Dis2 contained the expected I.4- and 4.3-lib HI'IIdlll fragments (Fig. 3) in addition to the 3.4-kb and 2.7-Itb fragments present in the recipient strain [362. Dis3 con- tained only the 2.7-kb DNA fragment. suggesting that genetic recombination between tandem copies of fus— IA resulted in the deletion of part of both copies of fax-IA plus the vector se- uences in between. The deletion was not due to a precise excision of pRZ2.8 because the 3.4-kb HiIIdll fragment was deleted and because fax-IA remained nonfunctional. The same filter was reprobed with ”P-labeled pBluescriptllSK(—) (data not shown). which hybridized to a 4.2-kb HI'IIdlII fragment. as expected. in Disl and Dis2 but not in Dis]. consistent with the hypothesized deletion event. The vector DNA hybridized to a 2.8-kb HiIIdlll fragment in all three disruptants. as expected. because of the integration of pSUlZ (which contains pUCl9) at the niuD locus. Southem hybridization analyses on identical TABLE 2. Sclerotium production in A. [xII-IIsI'IictIx . . Nani ‘ Av diam" Strain“ sclerotia" (gum) I362 till" 470 : IJII SUI (Afl‘) 2.400 470 z 90 Disl 3.000 490 : I40 DisZ lell 46" 1 tot) Disl 5.4th 460 : tot) ' 862 Is the recipient strain used In the [Iii- IA ldisruption etperiment SUI is an aflatoxin- -piiiduu'ng wi III-type. straI It. Disl. and -J an: three [as- M dis- ruptaiits selected for further study. canst oft two experime ulu I94 MAHANTI ET AL. A. Enoyl Reductase Mr...“ . r r: u t t I. 0L 0” no ‘ o—a u a a— 4. f... O r a. O ) S.e. In! A.. Ins-IA v F 'I-t r-—- 3° 2 z-: -r—- <-'¢ '. a- 11 r—- o- . 3 a: P l Se. test P 1 A4. tee-IA v . P--."'- :-a a- a -on‘ )—I no. on. ‘0.— 0". “—O a--- (on! X I'll-C G—n ‘-9 U )—I 3-? 0-3 an - Se. [at “‘0 3 2-2 hp. Ins-IA O—o tit—O g... .. O-B "INA 1—‘ .q—.. a—q O-o :— a r-— z-: x—r m— o ‘1'! S.e. last A..». Ins-IA . an >—- U—I- WI—Q D' 2-( ‘lI—Q )—-I 2 O—a 0.8 do 2-3 4"- <-~< S.c. In! An. tut-IA 0 0—3 1—-- ’ <..- M-. 1—-- -t—- x. r—- S.e. In! ‘ an! ’ ‘—‘ An. loo-IA D O =- 1- e F 8-3 2 '0-9 ‘fl—'§ (OI v-n < S.e. in! I ‘—§ O—Q - .- Pee- Ap. fat-IA O—o O—n I m—e ‘. S.e. In! <—¢ so-.i A4. tee-IA <-¢ 0-3 a p 1 S.e. fuel is Ly. tan-IA “—- Pan‘ 1' N 11 S.c. In! H A 81- B. MIP Transferase A4. Ina-IA It 8 -t—~ O—I <-< 1—q O 0—0 F ,n S.e. [at H 17 s l 5 A4. Ian-IA 0-. ”-0 <—~¢ ’0'“ ,-I O- O—u r-- 33. (at A... loo-IA a... 2—~ ‘eoa ,-. )-0 st—a S.e. (at A... tan-IA P I p SI I I 1. 53.1351 OTVTNVLNF KLQK ‘,u ‘=Zeo‘ yrnrvplt titraxitur 959f999 I|I|l== cscsosn O ) to: q - m m—o -r <-¢ IICIVOI lOSYSLL r t l L V X—r heltqple 0| 0 D I DSSSVSED 92 APPL. ENVIRON. MICROBIOL. on. C. . “no t ‘ . O-fl a-q O—n O—d O-G 1-2 2—2 0—3- Ill—O ) 8+ 2—: ‘—-§ ) r—- 0—8 0 no- uo‘ Z-i 1—-- :°_. 0- a o-a al.—an < "—‘ a—n <-¢ 3' M I I. X 1-IQ on. -<-~¢ ‘—‘I ‘- {-3 ‘-—‘ ,- no .4 0—0 m- o. a. x- I 1—‘ q—ao 5—0 0-0 -ug <-< al.—a. -ee‘ 'n—n ' . ‘ x' 3 a—~ aua‘ o-‘ ‘< . -0.‘ "" 2—~ r-- 1. :z-::<—< _N,_.Na. 3 a. a 3’ 1" :— ‘0 200‘ S—~ u.- .- x :‘00- V5 ”—a r-—- “-0 dlt mun. -t- I )—n - 4 a fill—- ) ‘6... -E:—: l' 9 0-0 3—3 0—3 ‘-‘B 2—~ 0' 3 ”I. “I 2.0 . <-< 0.. 3‘3 8 ‘- VAV a—- 40 r 65-: l-u a. <-< m—o -e. ‘ <-¢ z-a <—~< 8-3 2. o o—a o—a <—< <-¢ )—-n a—n O- 9 PH e a l ‘ P... 1133 FIG. 6. Gap (CCU) comparison of A, prmm'rr'rm fut-M and S. rrrn'itim- I545] products ( I5). Predicted amino acid sequences encoded in two regions ol [us-IA were compared with lunctional domains in the yeast I’ASI gene product by using the (KB soltwarc (iap. (A) (iap analysis ol the enoyl reductase lunctiunal domain. The [nutritive active-site motif is highlighted. (II) (Bap :tmtl)\t\ ot malonylpalmityl (M/l’) translerase lurwtional domain ’lhe putative active-site residue (serine) is highlighted Numbers in the yeast amino acid sequence are those reported by Kottig et al. (I5) Vertical lines between residues in the comparison represent identity. two dots represent more highly crmserved substitutions. and a single dot represents less highly conserved substitutimrs. genomic DNAs digested with Seal confirmed the llr'ndlll data (not shown). Sclerotium development. Disl and I)is2 produced four- to fivefold and Dis3 produced approximately ninclold more scle- rotia than I362 (Table 2). The number of sclerotia produced by the aflatoxin-producing strain SUI was similar to that pro- duced by Disl and Dis2; however. Dis3 produced twofold more sclerotia than SUI. DISCUSSION Because [as-IA is necessary for synthesis of NA. this argues that [as-IA encodes either noranthrone oxidase (III) or an activity involved in polyketide backbone synthesis. The large size of the [us-IA transcript suggested that it might encode a multifunctional protein. similar to pier/1 (7. 25). Nucleotide sequence analysis was conducted on two extensive regions of fax-IA to determine if predicted amino acid sequence data VOL. 62. I996 might provide clues about far-IA function (Fig. 6). Compari- son of the predicted amino acid sequence of the far-IA product with proteins in the GenBank and EMBL databases with the TFastA program detected a high level of identity with FASI proteins from Saccharomyces cerevisiae and Yanowr'a Iipolyrica (l5). FASI encodes the beta subunit of FAS. a protein which contains four functional domains typical of FASs. including (from amino terminus to carboxyl terminus) acetyltransferase. enoyl reductase. dehydratase. and malonyl/palmityl transferase (IS). A 435-amino—acid region in the [as-IA product displayed 40% identity and 58% similarity with the enoyl reductase do- main in FASI. while a l59-amino-acid region displayed 47% identity and 69% similarity to the malonyl/palmityl transferase domain (including the active-site residues). These two domains appeared in the same relative position and order in the [us-IA product as in FASI. These data strongly suggest that [as-IA encodes the beta subunit of a yeast-like FASI and support our new designation for this gene. [as-IA (formerly m'mx). Townsend et al. (22) proposed that hexanoatc, a six-carbon fatty acid. was the starting molecule for polyketide synthesis because NA. the first stable intermediate in AFBI synthesis. contains a six-carbon “tail" in which two kcto groups are com- pletely reduced to hydrocarbon. Hexanoatc was proposed to be extended to noranthrone. without further ketoreduction. by a PKS. Our data support this model; the firs-IA product is proposed to synthesize hexanoatc (or a similar fatty acid starter unit). while the pks/t product extends hexanoatc to noranthrone. Trail et al. (25) reported that the [us-IA transcript accumu- lates under aflatoxin-inducing conditions with the same pattern as nor-l and tier-I (20), suggesting that fax-IA. like nor-l and l't’f-I, is involved in secondary metabolism. Disruption of [as-IA in the current study had no apparent effect on the growth of A. [xrrrrsilicns on CZ. a defined minimal growth medium that contains no added fatty acids. Together. the data suggest that final/1 is involved in AFIII synthesis and not in the synthesis of fatty acids required for growth. Disruption of [03— IA also enhanced sclerotium development compared with the parental strain I362. a phenotype similar to pits/1 disruptants (25). Since no AFBI pathway intermediates accumulate in Disl. DisZ. or Dis3 or pier/1 disruptants. the accumulation of certain pathway intermediates (ie.. NA. AVN. and VA) appears to downregulate sclerotium development. This hypothesis is supported by previous observations. Strain CSlll (ver-l er-I pyrG), which accumulates VA (an interme- diate near the middle of the AFBI pathway). produces few sclerotia on CAM at 30°C (l9). Complementation of CSltl with ten] restores wild-type levels of AFBI synthesis and scle- rotium production. The nature of the interaction between AFBI synthesis and sclerotium development remains unclear and deserves further study. ACKNOWLEDGMENTS This work was supported by a cooperative research agreement with USDA and by grant CA52lltl3 from the National Cancer Institute. We thank Matthew Rarick for assistance with nucleotide sequence analysis. REFERENCES I. MI”. 1.. and II. I. Matelea. WM. Incorporation of labelled compounds into aflatoxins. Iliocbim. Biophys. Acta “MIR—420. 2. Armaler'atne. S. N. L M. De SM 5. WWII. and C. II. S. R. 'd ‘- '0 ‘0 2 . 22. J 4.0 25. 92a A. PARASITICUS AFLATOXIN BIOSYNTHESIS GENE far-IA I95 Bandaaatha. l969. Coconut as a medium for the experimental production of aflatoxin. Appl. Microbiol. Isms—04. . Ans-bet. F. M.. R. Brent. R. E. Klngston. I). D. Moore. J. G. Seldnian. J. A. Smith. and K. Struhl. I987. Current protocols in molecular biology. vol. I. John Wiley (1 Sons. Inc.. New York. . Bhatnagar. I)” K. C. Ehrlich. and T. E. Cleveland. I992. Oxidation-reduc- tion reactions in biosynthesis of secondary metabolites. p. 255—286. In "and- book of applied myuilogy. vol. 5. mycotoxins in ecological systems. Marcel Dehltet. New York. . Bhatnagar. IL. 8. I’. McCormick. I. S. Lee. and R. A. "III. l9ll7. ldentili- cation of O-methylsterigmatocystin as an allatoxin II, and (i. precursor in «Islam/Ins ,mrmin'r nr Appl. Environ. Microbiol. 53zlll3Il-Ill33, . Ilhatnagar. IL. (3. I’apne. J. E. Una. and T. E. Cleveland. IWS. Molecular biology to eliminate aflatoxins. Inform 5:2nZ-ZN. . Chang. I’.-I(.. J. W. Cary. J. Ya. I). Ilhatnagnr. and T. E. Cleveland. As- prrgrllm pumu'm‘m pk \A. a homolog of AslwrgiI/ns nirlnlnm WA. is required for allatoxiu II, biosynthesis. Mol. (ien. Genet. in press . Chang. I'.-K.. C. I). Skory. and J. Ii. Lina. IWZ. Cloning of a gene associated with allatoxin III biosynthesis in xliIx-rgillm lunmricm. ('urr. (ienet. 2|:23I- 233. . Cotty. I‘. J. IW. Atlatoxin and sclerotial production of Axpugrllm Ilium: influence of pll. l’hyropatlmlogy 732l25tl—l253. . IIritton. M. I'. was. lintymes and aflatoxin biosynthesis. Microbiol. Rev. 52274—295, . lhnracliova. I. I‘ll". Allatoxrns and human health. CRC Press. lloca Ralon. Fla. . lIanahan. I). I‘m}. Studies on transformation of En‘lmr‘rln'a rolr' with plas- mids. J. Mol. Biol. Innz557—SXII. . llorng. J.-S.. l’.-K. Chang. J. J. Pestka. and J. E. Una. I‘I‘Jtl. Development ol a homologous transformation system for Ava-millm ,amnilnm with the gene encoding nitrate reductase. Mol. (ien. (ienet. 224294-290. . Ilnrng. J.-S.. J. Ii. Lina. and J. J. I'estha. l‘lX‘l, Cloning and characteriration of the Iqu gene from an allatoxigenic strain of Ava-milk" pururnn'nr. Appl. Environ Mictiiliiol. SSQVII 73V“ . Knttlg. Il.. (I. Rottner. K. llecli. M. Schwelter. and I1. Sehwelter. l‘l‘ll Ilie [x'ntafunctional l-"ASI genes of Sm (humanism r'e‘n't'tttllt’ and l'nmim'n lipa- hiim are co-liuear and considerably longer than previously estimated. Mol. Gen. (Benet. Zlnzllltalll. . Lee. I- 8.. J. W. Ilennett. I. It. Goldblatt. and II. II. handln. W7". Nor- soloriuie acid from a mutant strain of Avx'rgrllm Immrnnm. J. Am. ()il Chem. Soc. Illz‘H—‘M . ”Int. S'.-ll.. and J. I}. ”III. l'l‘N. Structural and fundional characterization of the rev 'l genes and proteins associated with the conversion of versicolorin A to sterigmatocystin in aflatoxin biosynthesis. poster III. In Proceedings: Current Issues in l’ood Safety. Toxicology Center. Michigan State Univero sity. Iiast lgirising. Mich. . Manlatls. T.. Ii. If. I'ritsch. and J. Sarnhrnnlr. Will. Molecular cloning: a laboratory manual. Cold Spring Ilarbor laboratory. Cold Spring Ilarbor. N Y. . Slurry. C. ll. I‘nK. Chang. .I. Cary. and J. Ii. Una. "”2. Isolation and eharaeterimtion of a gene from Jinn-dim ,arrun‘rn‘nr associated with con- version ot versicolorin A to sterigmalmystin in allatoxin biosynthesis. Appl. linviron. MICttil‘itIl. 58:327-397. . Slurry. C. IL. I’.-K. Chang. and J. IE. Unz. IWJ. Regulated expression of the mm] and iw-I genes associated with aflatoxin biosynthesis. Appl. Iinviron. Miettrlviirl. 59:Ili-IZ—Ili-llr. Slurry. C. l).. J. S. Ilorng. J. J. Pestka. and J. E. Unz. I‘M). A transformation system for Aipmgillm puruumm based on a homologous gene involved in pyrimidine biosynthesis (pyrG). Appl, linviron. Microbiol. 56:13I5—332ll. Townsend. C. A.. S. M. McGuire. S. W. Ilmhst. T. I- (irayhlll. K. l‘al. and C. Ii. Ilarry III. I‘NI. laxamination of tetrahydro- and dihydrobisfuran lor- nration in aflatoxin biosynthesis: from whole cells to purilied enzymes. p. l4l— IS-l. In R. J. l'etroski and S. l’. McCormick (ed). Secrmdary-metabolite biosynthesis and metabolism. Plenum Press. New York. .. Trail. I-'.. l’.-K. Chang. J. W. Cary. and J. E. Lina. I‘I‘M. Structural and functional analysis ol the mm] gene involved in the biosynthesis of allatoxius by Avrrgrllm ,xnmuirm. Appl. l'nviron. Microbiol. 60:4tl7Il—«1lltl5. . Trail. II. N. Mahantl. and J. E. Lina. I005. Review—molecular biology ol aflatoxin biosynthesis. Microbiology HINSS-WiS. Trail. I-'.. N. Mahantl. M. Rariclr. R. Mehigh. S.-II. Liang. R. Zhou. and J. Ii. Unix IWS. A physical and transcriptional map of an aflatoxin gene cluster in Arlrrgrllm purrm'rn'm and the functional disruption of a gene involved early in the aflatoxin pathway. Appl. linviron. Microliiol. 6|:2Hi5-2fr73. Ya. J.. I’.-K. Chang. J. W. Car-y. M. Wright. I). Ilhatnagar. 'I'. I5. Cleveland. (I. A. Payne. and J. E. lan. IWS. Comparative mapping of aflatoxin pathway gene clusters in Auriga/Ins Immu’nrni and Ava-milk" Ilium. Appl. Environ. Microbiol. 6|:23h5—237l. APPENDIX B Appendix B - Sequence analysis of the A. parasiticus pyrG The absence of a sexual cycle in certain species has made the production of mutants and subsequent isolation of genes a diflicult prospect. Several Aspergillus spp. including A. parasiticus and A. flavus fall into this category. Fortunately, transformation systems have been developed which allow the introduction of naked DNA to 1) complement mutations of interest and 2) restore the fimction of a marker gene. Two "E common marker systems use the complementation of mutant genes encoding nitrate reductase (niaD) (Wu and Linz, 1993), and orotidine 5'-monophosphate decarboxylase : (pyrG). The pryG is responsible for the conversion of orotidine 5'-monophosphate to L uridine monophosphate during pyrimidine biosynthesis (Skory, 1992). Auxotrophic strains require media supplemented with uridine to grow. Without it genomic replication cannot proceed. Transformation of these strains with a functional pyrG results in conversion to prototrophy. This particular marker system is useful because it is eflicient. Skory reported that he was able to obtain 30 to 50 stable transformants per ug DNA with no background growth (Skory et al, 1990). This transformation system has been used in many species including A. nidulans (Oakley et al, 1987), A. niger (Wilson et al, 1988), A. flavus (Woloshuk et al, 1989), A. parasiticus (Skory et a1, 1990), S. cerevisiae (Rose et al, 1984), Neuropora crassa (Glazebrook et al, 1987), and Penicillium chrysogenum (Cantoral et al, 1988). The nucleotide sequences of pyrG from each of these species have been published with the exceptions of A. flaws and A. parasiticus. By sequencing the third clone of the cosmid NorA (by DNA Technologies) 2700 base pairs of A. parasiticus pyrG was determined (Figure 19). Subsequent alignment of the pyrG genes at the amino 93 94 acid level is presented in Figure 20. The identity between A. parasiticuspyrG and those from the other species ranged from 78% with A. nidulans down to 38% with N. crassa. Also, the proposed intron common to all of the published pyrG sequences appears to be present in this sequence as well. 95 Figure 19. Nucleotide sequence of the A. parasiticus pyrG. 96 .2 as; ¢U¢OBO¢¢¢¢OB¢OFOOOUBH¢O O .— '— H. > > H. O DFVVFTTGVN HMTPGCK NC STTEEEAOAD 99 <<<<<<< DWWXKWX Figure 20. APPENDIX C Appendix C - Protocols used in promoter studies The identification of regulator genes such as afl-R is one key in the successful utilization of a genetic approach for eliminating aflatoxins from the food chain. This, however, is only part of the battle. It is necessary to identify how the regulators work. Do they act in a positive or negative manner? Do they act at one site or several? Because the aflatoxin pathway is regulated at least in part at the level of transcription it is reasonable to assume that there are regulatory factors involved in some aspect of transcription. It may also be safe to say that this regulation occurs at the promoter region of one or more genes in the pathway. Studies investigating the proteins that bind promoters of known aflatoxin genes may identify key regulators of the pathway. These studies not only would attemp to identify the factors involved but they would also define the specific sequences they bind. To do these kinds of studies the polymerase chain reaction, nuclear extraction of proteins, gel retardation, and DNAase foot printing, will be employed. The following are modified procedures for performing the above procedures with the exception of DNAase foot printing. El l' [H I R |° l] I .1! .,. Purpose: Extract proteins from the nucleus of Aspergillus parasiticus including possible transcription factors involved in aflatoxin biosynthesis. Modified from Timberlake (1986) and Nagata et a1 (1993). 100 101 Materials All materials should be autoclaved and/or treated as indicated prior to use. 250ml GSA bottles 8834 tubes Spatula and/or rubber policeman 10ml pipettes 4L filter flask(s) Buchner fimnels (1 or 2 for a 4L flask, 1 or 2 for a 1L flask) Glass beads Mira cloth Cheese cloth The followingreagents should be stored at 4°C or kept on ice while being used. Distilled water 10XSSE salts: 1.0M KCl, 0.1M EDTA, 0.1M Tris base. Make stock of this and autoclave. Just prior to using add Sperrnidine to 0.04M and Spermine to 0.04M. Adjust pH to 7 with HCl. 1.0XSSE salts (made just prior to use): Combine 100ml of 10XSSE salts with lml B- mercaptoethanol, 171.2g Sucrose, and 10ml of PMSF (0. 1M in 95% ETOH). Bring to one liter. Nuclear Extraction Bufl'er: 10% glycerol, 0.015M Hepes-KOH (pH7.9), 0.5M KCl, 0.005M MgC12, 0.5mM EDTA. Make a stock of this and autoclave. Just prior to using add 0.001M DTT, 0.5mM PMSF, and two of the following (to IOug/ml each) antipain, chymostatin, leupeptin, and/or pepstatin. 102 Dialysis Bufi‘er (prepare just prior to using): 15% glycerol, 0.015M Hepes-KOH (pH7.9), 0.1M KCl, 0.001M EDTA, 0.002M DTT, 0.5mM PMSF, and two of the following (to long/ml each) antipain, chymostatin, leupeptin, and/or pepstatin. Method 1. Inoculate four 1L flasks containing 500ml medium’ with 5x10“ spores each. 2. Incubate the flasks 46 hours in the dark at 29°C while shaking at 150 rpm. 3. Harvest the mycelia by vacuum filtration through one layer of mira cloth in a 4L Buchner funnel. Wash the mycelia with chilled water and vacuum dry. 4. Remove the mycelia fi'om the 4L Buchner firnnel and place into a 250ml GSA bottle. 5. Add 1.0XSSE and mix with a spatula or rubber policeman until a slurry is formed. 6. Using the bead beater. Fill the bead beater container half to two thirds firll with glass beads’. The actual amount depends on the volume of the slurry. The more the slurry the less the beads (note: the container must be at least half full of beads). Add the slurry. Add 1.0XS SE until the bead beater container is full. Insert blades and secure with collar. Add ice to the collar and blend as follows: blend 45 sec, set 30 sec, blend 45 sec, set 30 sec, blend 30 sec, set until beads settle to bottom of container. 7. Gravity filter the slurry using a 1L Buchner firnnel containing four layers of mira cloth covered by two layers of cheese cloth°. The supernatant should be collected in a 250ml GSA bottle which is surrounded by ice. To speed up this filtration two 1L Buchner fimnels may be used. 8. Rinse beads in the bead beater container. Add 1.0XSSE to the bead beater container. Insert blades and secure with collar (ice is not necessary). Run bead beater for five to 10 seconds. Let the beads settle. Add the slurry to the IL Buchner funnel(s) from the 103 previous step. 9. Centrifuge the GSA bottle(s) at 8000rpm (using the GSA rotor) for 20 minutes at 4°C. 10. Discard the supernatant. Resupend the pellet in 30ml 1.0XSSE using a spatula or rubber policeman. 11. Transfer the suspension to Oakridge tubes (S 834 tubes). Centrifuge at 8200rpm for 20 minutes at 4°C. The point of this centrifiagation is to obtain a pellet and a clear supernatant by repeating this step as many times as it takes. It may be more correct to say that performing this centrifugation a total of three times is best. This is due to the fact that sometimes the first centrifugation will yield a clear supernatant, whereas other times a clear supernatant is not obtained even afier four centrifiigations. Because of these inconsistencies and trial and error three times should suffice. After each centrifugation decant the supernatant and resuspend the pellet in 1.0XS SE. 12. Resuspend pellet in five to 10ml of nuclear extraction buffer. Put tubes in ice and rock on platform shaker for 30 minutes. 13. Transfer suspension to ultracentrifiige tubes (for rotor T8651). Centrifuge at 36,500rpm for 60 minutes. 14. Decant the supernatant to 15ml conical centrifiige tubes by puncturing the ultracentrifiige tubes with a high gauge needle. To get the entire supernatant out of the tube it may be necessary to carefiilly (so as not to disturb the pellet) cut the top of the tube ofl‘ with a pair of scissors. 15. Transfer the extract to dialysis tubing (molecular weight cutofl‘ 3500). 16. Dialyze in dialysis bufi‘er overnight. 17. Decant tubing into 8834 tubes. Centrifilge at 11,200rpm to pellet debris 104 (clarification). 18. Transfer supernatant to a Centriprep 10 concentrator (Amicon). The instructions with these concentrators say to centrifirge approximately 45 minutes. This does not take into account the temperature (4°C) and the glycerol content of this sample. Because of the lower temperature and higher glycerol content used in this procedure the times will be tremendously increased. This concentrator is only capable of handling 15ml at a time. You will need to centrifuge using a GSA rotor at 43 00rpm (3 000xg) for a couple hours. At that time add more of the extract and continue centrifugation. Continue to do this until all of the extract has been added. Centrifuge until approximately one or two ml of extract remains. This may take an overnight centrifirgation. l9. Determine the concentration of the extract using the protein concentration assay from Biorad. 20. Store extracts at -80°C. ’Possible media are Glucose Minimal Salts (GMS) used to induce aflatoxin production. Peptone Minimal Salts (PMS) used to supress aflatoxin production. Nitrate Minimal Salts (NMS) used to delay aflatoxin production. NMS is the same as GMS except the sole nitrogen source is NaNO3. *The glass beads are prepared by soaking in 0.5M HCl (to clean) then rinsing with deionized water (to remove HCl). The beads are ready to be dried when the pH of the rinse water reaches approximately 7.0. °The mira cloth and the cheese cloth used in this step are prepared by boiling in EDT A three times followed by boiling in distilled water four times then dried. 105 I I CI . R |° Purpose: Amplify specific segments of DNA which are suspected of serving as promoter regions for aflatoxin genes. The following is a list of reagents used in the reaction mixture for PCR. Reagent Volume/sample DEPC water (To SOuL) MgCl2 (25mM) 6uL lOXPCR buffer II SuL dNTP mix (lOmM) 4pL Primer 1 (200ng/uL) luL Primer 2 (200ng/uL) luL Taq Polymerase (5units/uL) lpL DNA template (200ng) ? Total volume SOuL These samples are then placed in a therrnocycler (Perkin Elmer) and subject to the following conditions. -Hold at 95°C for five minutes. -Cycle the samples 30 times through 95 °C for one minute 60°C for one minute 72°C for three minutes -Hold at 72°C for 10 minutes -Hold at 4°C until removed 106 (This may be replaced by Qiagen method) The products are then electrophoresed on a 0.8% agarose gel (containing ethidium bromide) followed by excision of the bands and subsequent purification. These purified products are end labelled using the Ready-to-go labelling kit (Pharrnacia Biotech) and y-"P ATP. Finally, these products are used in mobility shift DNA-binding assays. Ml'l'l Sl'fllDHl-B' l' l 11. GIEI I l . Purpose: To determine if a fragment of DNA is bound by protein(s) from a nuclear protein extract. From Current Protocols (Ausubel et al, 1987). REFERENCES List of References Ausubel, F.M., R. Brent, R.E. Kingstone, D.D. Moore, J.G. Seidman, J.A. Smith, and K. 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