Phanerochaete chrysosporium

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1 APPLED AND ENVRONMENTAL MCROBOLOGY, Oct. 1982, p Vol. 44, No /82/ $02.00/0 Copyright 1982, American Society for Microbiology solation and Complementation Studies of Auxotrophic Mutants of the Lignin-Degrading Basidiomycete Phanerochaete chrysosporium MCHAEL H. GOLD,* THERESE M. CHENG, AND MARY B. MAYFELD Department of Chemistry and Biochemical Sciences, The Oregon Graduate Center, Beaverton, Oregon Received 3 May 1982/Accepted 18 June 1982 A variety of auxotrophic strains of Phanerochaete chrysosporium were isolated after treatment of conidia with UV and X rays. Complementation studies with these strains demonstrated heterokaryotic mycelia and conidia in this organism. Nuclear staining also showed that conidia can be mono-, di-, or multinucleate. Complementation tests allowed the separation of each auxotrophic class with the same phenotype into complementation groups. The basidiomycete Phanerochaete chrysosporium and other white rot species have potential application in a variety of schemes for the commercial processing of lignocellulose. These organisms have already been used in numerous studies concerned with lignin degradation (2), cellulose degradation (4), and lignocellulose bioprocessing applications (10). The realization of these potentials would be considerably enhanced, however, if genetic methods for selecting strains with superior capacities were available. To date no reports on the isolation of genetic marker strains, on complementation, or on recombination with this potentially valuable organism have appeared. n a previous publication (6) we described methods for inducing colonial growth and for replica plating with this organism, which we predicted would prove to be useful for the isolation of mutant strains. n a subsequent publication (7) we elucidated the physiological conditions required for fruit body formation in this organism. n this report we describe methods for mutagenesis of P. chrysosporium conidia and subsequent isolation and characterization of a variety of auxotrophic marker strains. We also describe studies on complementation with these mutant strains. The complementation studies indicate that heterokaryon formation is readily accomplished and that the heterokaryotic state can pass through the conidial stage of growth. Cytological studies confirming the presence of mono-, di-, and multinucleate conidia are also described. Organism. A culture of P. chrysosporium ME446, obtained from the U.S. Forest Products Laboratory, Madison, Wis., was maintained on slants as previously described (6). After 7 days of growth, conidia were washed from slants, filtered through glass wool, and diluted in distilled water. The number of conidia in suspension was determined with a hemacytometer. Mutagenic treatments. UV mutagenesis was performed by using a modification of a previously described procedure (3). Stirred suspensions of conidia (2 x 106/ml) in 10 ml of water were irradiated at 25 C in petri dishes under subdued light. A 15-W General Electric germicidal lamp was used at a distance of 15 cm to irradiate the conidia for 1.5 min. After this exposure, approximately 10% of the spores remained viable. Mutagenesis with X rays was also performed by using a modification of a previously described procedure (3). Stirred suspensions of conidia (2 x 106/ml) in 10 ml of water were irradiated for 80 min at a distance of 15 cm. The total dose was 35,000 rads. After this exposure, approximately 10% of the spores remained viable Ȧfter mutagenesis, spores were diluted appropriately and plated onto solid medium containing Vogel medium N (14) with thiamin replacing biotin (modified Vogel medium). Sorbose (1%) and sodium deoxycholate (0.01%) were added to induce colonial growth (6), and the medium was supplemented with yeast extract (0.02%), hydrolyzed casein (type 1 from milk) (0.2%), and a vitamin mixture as previously described (6). Cultures were incubated at 28 C unless indicated otherwise. Master plates containing complete medium which had been incubated at 28 C for 7 days (allowing time for colony formation and conidiation) were replica plated onto minimal medium as previously described (6). The master plates were then stored at 4 C, whereas the replicate plates were incubated at 28 C for 5 to 7 days and then visually compared with the master plates. Colonies which grew on complete medi- 996

2 VOL. 44, 1982 NOTES 997 TABLE 1. Phanerochaete chrysosporium auxotrophic mutants: isolation and classification into complementation groups Mutant (isolation no.) 1 Amino acid-requiring strains Leul Leu2 Leu3 Leu4 Leu5 Argl Arg2 Arg3 Arg4 Arg5 Hisl His2 His3 His4 leu-vall leu-val2 leu-val3 Metl Met2 Met3 Met or Cysl Met or Cys2 Met or Cys3 Met or Cys4 Cysl Lysl Vitamin-requiring strains Nicl Ribl Rib2 Adenine-requiring strains Adel Ade2 Ade3 Ade4 (OGC 708-1) (OGC 731-4) (OGC 923-1) (OGC 923-3) (OGC 923-4) (OGC 112-1) (OGC 128-1) (OGC 702-2) (OGC 708-3) (OGC 731-5) (OGC 413-4) (OGC 504-2) (OGC 708-4) (OGC 923-2) (OGC 119-1) (OGC 128-4) (OGC 731-3) (OGC 213-2) (OGC 115-1) (OGC 107-4) (OGC 128-3) (OGC 128-4) (OGC 413-3) (OGC 708-8) (OGC 912-1) (OGC 325-2) (OGC ) (OGC 103-1) (OGC 923-6) (OGC 107-1) (OGC 128-2) (OGC 702-4) (OGC 923-5) Group V Complementation groups Mutants in group Leu2, Leu4 Leul, Leu5 Leu3 Argl, Arg3, Arg4, Arg5 Arg2 Hisl His2 His3 His4 leu-vall, leu-va2 leu-val3 Metl Met2 Met3 Met or Cysl, Met or Cys2, Met or Cys3 Met or Cys4 Ribl Rib2 Adel Ade2 Ade3, Ade4 um but not on minimal medium were retested on slants to determine the specific supplement required. n some cases, to quantify the effect of supplements on the growth of various mutant strains, the strains were grown in liquid culture. Cultures were grown in 50 ml of modified Vogel medium, containing 1% glucose and supplements as indicated. Cells were grown in Erlenmeyer flasks on a New Brunswick G-10 shaker operating at a speed of 150 rpm and describing a 5-cm-diameter circle. After 48 to 96 h, the mycelial mat was filtered, washed, dried, and weighed. Complementation studies. Mycelia from any two of various auxotrophic mutants were inoculated onto solid minimal medium to force complementation. Conidia produced by the resultant heterokaryotic mycelium were collected as described above, filtered, diluted, and plated onto

3 998 NOTES APPL. ENVRON. MCROBOL. FG. 1. Heterokaryon formation in Phanerochaete chrysosporium. Wild-type conidia (A), a mixture of Adel and Argl conidia (B), and conidia obtained from an Adel-Argl heterokaryon (C) were plated onto minimal medium containing colony-inducing agents and incubated for 8 days at 28 C. solid minimal medium containing colony-inducing agents. At the same time the conidia from single or mixtures of auxotrophic mutants were also plated onto minimal medium. Cytological quantification of nuclei in conidia. Mycelia from various strains were grown on malt extract agar (9) consisting of 2% malt extract, 2% glucose, 0.1% bactopeptone, and 1.5% agar, and conidia were harvested as previously described (6). Under these conditions, 8- day-old mycelia from the wild-type organism give a high percentage of mononucleate conidia (personal communication, E. N. Davis, U.S. Department of Agriculture, Peoria, ll.). The procedures used to attach and fix the spores to a glass cover slip by using Helly fluid and to stain the nuclei by using Giemsa-hydrochloride were essentially as described previously (12, 13). UV radiation used under the conditions described above resulted in a very low incidence of auxotrophic mutants recovered. Less than 1 mutant was found per 10,000 colonies screened. With X-ray irradiation, approximately 1 mutant per 1,000 colonies screened was recovered, or approximately 1 mutant per 35 plates. Attempts at filtration enrichment (15) of the mutants were unsuccessful, apparently due to the rapid clumping of ungerminated spores with each other and with growing mycelia in shaking cultures. The following 33 mutants (Table 1) were isolated and identified: 5 requiring leucine (Leu-), 5 requiring arginine (Arg-), 4 requiring histidine (His-), 3 requiring isoleucine-valine (leu-val-), 3 requiring methionine (Met-), 4 requiring methionine or cysteine (Met or Cys-), 1 requiring cysteine (Cys-), 1 requiring lysine (Lys-), 1 requiring nicotinamide (Nic-), 2 requiring riboflavin (Rib-), and 4 requiring adenine (Ade-). Each of these mutants was tested in separate tubes containing modified Vogel medium (14) and glucose to determine the effect of a variety of supplements. A culture of each mutant strain was tested to ensure that the phenotype persisted through conidiation and subsequent germination of isolated colonies. Ribl, Cysl, Met or Cysl, and Metl were the only UV mutants found. The mutants isolated were not randomly distributed with respect to phenotype. Four of the mutants required histidine, and an additional five mutants required leucine, whereas mutants requiring several other amino acids were unrepresented. Mutants requiring both isoleucine and valine have been isolated previously with Neurospora crassa (5). When any two of the mutants listed in Table 1 requiring different supplements were inoculated together onto minimal medium, vigorous growth ensued after a short lag time, suggesting that heterokaryon formation occurred, although cross feeding could not be ruled out. To differentiate between these two alternatives, two mutants, Argl and Adel, were allowed to form a presumed heterokaryotic mycelium on minimal medium. Conidia derived from this mycelium were carefully filtered through glass wool several times to remove traces of mycelium, diluted, and finally plated onto minimal medium containing colony-inducing agents. Figure 1 shows the results of such an experiment. Colonies formed from wild-type conidia (Fig. 1A), colonies formed from conidia of the Argl-Adel heterokaryon (Fig. 1C), and a dish which had been incubated with a mixture of Adel and Argl conidia (total spores) (Fig. 1B) are shown. These results demonstrated that heterokaryon formation does occur in the vegetative mycelia stage. They also demonstrated that the heterokaryotic state can pass through the conidial stage and suggested that the conidia can be at least dinucleate in morphology.

4 VOL. 44, 1982 TABLE 2. Number of nuclei per conidiospore in the wild type and a variety of auxotrophic and heterokaryotic strains of Phanerochaete chrysosporiuma Strain Mono- % % Multinucleate Dinucleate nucleate' Wild type Auxotrophic mutants Nicl Ribl Metl Adel Argl Heterokaryons Nicl-Ribl Adel-Nicl Metl-Adel Adel-Ribl 1 99 Adel-Argl a Conidia were fixed to cover slips and stained with Giemsa as described in the text. Most of the multinucleate conidia were trinucleate. Specific complementation tests (5) were also conducted with all combinations of individuals from each class of auxotroph. These tests allowed the separation of each auxotrophic class into complementation groups (those mutants which did not complement with one another). The number of complementation groups distinguished and the members of each group for each auxotrophic class are also listed in Table 1. The demonstration that the heterokaryotic state can pass through the conidial stage suggested that the conidia of heterokaryons could be dinucleate. To test this hypothesis, populations of conidia isolated from cultures of the wild type, of several auxotrophic mutants, and of heterokaryons were stained as described above. The number of nuclei in each of at least 100 stained conidia were then recorded. The results of this experiment are shown in Table 2. Under the culture conditions used, approximately 62% of the wild-type conidia were mononucleate, 37% were dinucleate, and 1% were multinucleate. The percentage of mononucleate conidia found in the auxotrophic mutants grown under identical conditions with the addition of supplements varied from 8 to 16%, whereas the percentage of dinucleate conidia varied from 79 to 94%. Of the heterokaryons tested, the percentage of mononucleate conidia varied from 1 to 10%, whereas the percentage of dinucleate conidia varied from 89 to 99%. When conidia from the Adel-Nicl heterokaryon were plated onto minimal medium and NOTES 999 subsequently transferred to supplemented medium, some Ade- and Nic- single mutants were recovered. The recovery of these original strains from this heterokaryon is additional proof that heterokaryosis had occurred. n a previous study (6) we developed a medium which induces colonial growth and allows replica plating of the lignin-degrading fungus P. chrysosporium. n this report we describe the use of those techniques for the isolation of auxotrophic mutants of this organism. Although UV and X rays were used to mutagenize conidia from this organism, under the conditions used, X rays yielded many more mutants and were used for most of the experiments. The low recovery of mutants may be partially explained by the high incidence of dinucleate conidia, although the low percentage of postmutagenesisviable conidia probably ensures that the surviving conidia are effectively mononucleate (3). As described above, the auxotrophic mutants obtained were not completely random with respect to phenotype. Whereas five Leu- mutants were obtained, mutants requiring either phenylalanine, tryrosine, or proline were not. This is probably a result of the nonspecific selection method employed. Hydrolyzed casein was used as a source of all amino acids except tryptophan. This nonspecific plating medium may lead to inhibition of the growth of certain types of mutants (1). n addition, it has been previously reported (8, 11) that certain auxotrophic mutants inhibit the growth of other auxotrophic and prototrophic strains. This phenomenon would also lead to the nonrandom isolation of auxotrophs as found here. To minimize these problems, experiments are planned on the use of individual supplements for the isolation of specific mutants. The relatively small number of vitamin-requiring mutants isolated is probably due to the ability of these mutants to grow on trace amounts of supplements which may leak from surrounding prototrophic strains. Complementation tests with these strains indicated that P. chrysosporium can readily form a heterokaryotic mycelium and that the heterokaryotic state can pass through the conidial stage. Each of the auxotrophic groups could be further classified into at least two complementation groups. This result indicates that many of the auxotrophs isolated are nonallelic, suggesting that the nonrandom isolation of mutants is probably not due to especially susceptible loci (hot spots) in the genome. n the case of Arg complementation group, however, this cannot be ruled out. The results of the complementation tests indicate that these auxotrophic mutants are recessive. Cytological studies indicated that both the wild-type and the heterokaryotic mycelium

5 1000 NOTES could give rise to dinucleate as well as mononucleate conidia. This is consistent with our complementation results which indicated that the heterokaryotic state passes through the conidial stage. The wild-type conidia were 60% mononucleate, whereas the conidia from heterokaryotic mycelia ranged from 1 to 10% mononucleate. The physiological significance, if any, of this result remains to be tested. Studies on crossing and recombination with the mutants that we have isolated are in progress. Additional studies, using several of these mutants to understand the regulation of lignin metabolism, are planned. We thank Alberta Herman, U.S. Department of Agriculture, Peoria, ll., for helpful suggestions on nuclear staining. This work was supported by the Crown Zellerbach Co. and the Weyerhaeuser Co. and by grant PCM from the National Science Foundation. LTERATURE CTED 1. Auerbach, C., and B. J. Kilbey Mutation in eukaryotes. Annu. Rev. Genet. 5: Crawford, R. L Lignin biodegradation and transformation. Wiley-nterscience, New York. 3. Davis, R. H., and F. J. DeSerres Genetic and microbiological techniques for Neurospora crassa. Methods Enzymol. 17A: Erlksson, K.-E Cellulases of fungi, p n A. APPL. ENVRON. MCROBOL. Hollaender and R. Rabson (ed.), Trends in the biology of fermentation for fuels and chemicals. Plenum Publishing Corp., New York. 5. Fincham, F. R. S., and P. R. Day Fungal genetics. Blackwell Scientific Publications, Ltd., Oxford, England. 6. Gold, M. H., and T. M. Cheng nduction of colonial growth and replica plating of the white rot basidiomycete Phanerochaete chrysosporium. Appl. Environ. Microbiol. 35: Gold, M. H., and T. M. Cheng Conditions for fruit body formation in the white rot basidiomycete Phanerochaete chrysosporium. Arch. Microbiol. 121: Grigs, G. W Back mutation assay method in microorganisms. Nature (London) 169: Haynes, W. C., L. J. Wikerham, and C. W. Hesseltine Maintenance of cultures of industrially important microorganisms. Appl. Microbiol. 3: Kirk, T. K., and H.-M. Chang Potential applications of bioligninolytic systems. Enzyme Microb. Technol. 3: Pontecorvo, G. C., J. A. Roper, L. M. Hemmons, K. D. MacDonald, and A. W. J. Bufton The genetics of Aspergillus nidulans. Adv. Genet. 5: Robinow, C. F Mitosis in the yeast Lipomyces lipofer. J. Biophys. Biochem. Cytol. 9: Robinow, C. F., and J. Marak A fiber apparatus in the nucleus of the yeast cell. J. Cell Biol. 29: Vogel, H. J Distribution of lysine pathways among fungi: evolutionary implications. Am. Nat. 98: Woodward, V. W., J. R. DeZeeuw, and A. M. Srb The separation and isolation of particular mutants of Neurospora by differential germination of conidia, followed by filtration. Proc. Natl. Acad. Sci. U.S.A. 40: Downloaded from on October 6, 2018 by guest