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BRIEF REPORT Folia Mierobiol. 82, 177--180 (1987) Genetic Recombination in Auxotrophic of Phanerochaete chrysosporium It. KR~J~i Department of Genetics, Microbiology a~rtd Biophysics, l~aculty of Sciences, Charles University, 128 4g Prague 2 Strains t~eceived December 16, 1985 AB,~TRACT. Four auxotrophie strains of the ligninolytie basidiomycete Phat~erochaete chrysospo- riura were obtained by VV mutagenesis. The heterokaryotie mycelium formed by complemen- ration of different auxotrophie isolates was able ~o fruit and produce basidiospores. From the basidiospore progeny of the heterokaryons prototrophic strains and strains with a recombined set of parental nutritionM requirements were isolated. Genetic recombination hence takes place in fruit bodies pro(tueed by the heterokaryotie myeelium. The basidiomycete Phanerochaete chrysosporium and other lignin-degrading microbial species have their potential application in the microbial treatment of lignin and lignocellulose. For the utilization of this fact it is necessary to clarify the physiology and molecular mechanisms of biodegradations as well as to develop techniques of genetic manipulation for the construction of industrially applicable strains. Thus, for instance, protoplast fusion (Pettey and Crawford 1984) and classical techniques, i.e. mutation and selection (Johnsrud and Eriksson 1985), were applied in this area. Genetics of P. chrysosporium is only little known at present. As a new species it was described by Burdsall and Eslyn (1974) who suggested that a perfect stage of ~porotrichum pulverulentum NOVOBRA~OVA is probably involved. Both organisms were compared at the DNA level by Raeder and Broda (1984). Their close relatedness was confirmed by means of DNA hybridization. At present the two species are considered to be identical. Papers studying the life cycle of P. chrysosporium in detail have not yet been published but it is known that only conidiospores are produced during cultivation on agar media with glucose. The formation of fruit bodies and basidiospores can be induced by cultivation in a medium with cellulose, thiamine and with a low nitrogen content (Gold and Cheng 1979). A series of papers by Gold and Cheng (1978, 1979) and Gold et al. (1982, 1988) were devoted to the methods of genetic manipulation. As an article concerning the recombination of auxotrophic markers in this species has recently been

Genetic recombination in auxotrophic strains of Phanerochaete chrysosporium

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Page 1: Genetic recombination in auxotrophic strains of  Phanerochaete chrysosporium

BRIEF R E P O R T

Folia Mierobiol. 82, 177--180 (1987)

Genetic Recombination in Auxotrophic of Phanerochaete chrysosporium

It. KR~J~i

Department of Genetics, Microbiology a~rtd Biophysics, l~aculty of Sciences, Charles University, 128 4g Prague 2

Strains

t~eceived December 16, 1985

AB,~TRACT. Four auxotrophie strains of the ligninolytie basidiomycete Phat~erochaete chrysospo- riura were obtained by VV mutagenesis. The heterokaryotie mycelium formed by complemen- ration of different auxotrophie isolates was able ~o fruit and produce basidiospores. From the basidiospore progeny of the heterokaryons prototrophic strains and strains with a recombined set of parental nutritionM requirements were isolated. Genetic recombination hence takes place in fruit bodies pro(tueed by the heterokaryotie myeelium.

The basidiomycete Phanerochaete chrysosporium and other lignin-degrading microbial species have their potential application in the microbial treatment of lignin and lignocellulose. For the utilization of this fact it is necessary to clarify the physiology and molecular mechanisms of biodegradations as well as to develop techniques of genetic manipulation for the construction of industrially applicable strains. Thus, for instance, protoplast fusion (Pettey and Crawford 1984) and classical techniques, i.e. mutation and selection (Johnsrud and Eriksson 1985), were applied in this area.

Genetics of P. chrysosporium is only little known at present. As a new species it was described by Burdsall and Eslyn (1974) who suggested that a perfect stage of ~porotrichum pulverulentum NOVOBRA~OVA is probably involved. Both organisms were compared at the DNA level by Raeder and Broda (1984). Their close relatedness was confirmed by means of DNA hybridization. At present the two species are considered to be identical. Papers studying the life cycle of P. chrysosporium in detail have not yet been published but it is known that only conidiospores are produced during cultivation on agar media with glucose. The formation of fruit bodies and basidiospores can be induced by cultivation in a medium with cellulose, thiamine and with a low nitrogen content (Gold and Cheng 1979). A series of papers by Gold and Cheng (1978, 1979) and Gold et al. (1982, 1988) were devoted to the methods of genetic manipulation. As an article concerning the recombination of auxotrophic markers in this species has recently been

Page 2: Genetic recombination in auxotrophic strains of  Phanerochaete chrysosporium

| 7 8 R. KREJ(~:[ Vol 32

published (Alic and Gold 1985) we present here our experimental results obtained in this area.

Phanerochaete chrysosporium, strain 284 B (wild type) was kindly supplied b y Dr. Setliff (ForinteIc Corp., Vancouver, Canada). All strains were main- tained on complete agar slants (Snider and Raper 1958) at 4 ~ They were cult ivated in a thermostat at 28 ~ unless otherwise stated. A minimal me- dium of the following composition was used (g/L): MgS04.7H20 0.5, KH2PO4 0.6, K2HP04 0.4, (NI-I4)2SOa 0.5, glucose 20, agar 20.

TABLE I. Isola ted auxotrophie m u t a n t s

Mutan t Growth requirement.

Met 21 methionine Ado 21 adenine Nic21 nicotinic acid Sr21 methionine, cysteine, thiosulfate

For the mutagenesis conidia of the wild strain were washed from the agar slant with 0.9 ~o NaCl solution, filtered through cotton and counted in a hemacytometer . Ten mL of the conidial suspension (spore concentration 107/mL) were irradiated with UV light (15 W lamp, distance 230 mm, 20 ~ with permanent mixing for 20 min and then placed in the dark at 4 ~ for 70 min (Eriksson and Johnsrud 1983). The survival ratio was about 0.02 % under these conditions. The layer-plating technique (Reaume and Tatum 1949) was used for the isolation of auxotrophic strains. Irradiated conidiospores were spread on the minimal medium containing 10 g of L- sorbose and 0.1 g sodium deoxyeholate per L instead of glucose, in order to induce the colonial growth (Gold and Cheng 1978). After a 7-d incubation the positions of growing colonies were marked and the plates were overlayered with 5 mL of a complete sorbose medium cooled to 38 ~ and containing (g/L): MgSO4.7H20 0.5, KH2PO4 0.6, K2HPO4 0.4, ammonium tar trate 0.5, thiamine 5 mg/L, sodium deoxyeholate 0.1, yeast extract 2, casein hydrolyzate 1, L-sorbose 10, agar 20. After an additional 3-4-d incubation the newly grown colonies were isolated and their nutritional requirement was identified. By means of this technique 4 auxotrophic strains were obtained (Table I). A single auxotrophic isolate was detected among about 2000 surviving colonies.

Two different auxotrophic strains were inoculated in a close proximity on the complete medium and cult ivated for 24 h in order to improve the

TABLE I I . Crosses single m u t a n t • single m u t a n t

Cross P ro to t rophs Pa ren ta l auxo t rophs Double Total m u t a n t s

Ade21 • :Nie21 Nic21 • Met21 Ade21 • Met21

22 32 Ade - 32 57ic- 19 105 38 48 Nic- 89 Met- 45 220 10 72 Ade - 117 Met- 12 211

Page 3: Genetic recombination in auxotrophic strains of  Phanerochaete chrysosporium

1 9 8 7 R E C O M B I N A T I O N I N P. chrysosporium 177

T A B L E I I I . C r o s s d o u b l e m u t a n t x s i n g l e m u t a n t

P a r e n t a l s t r a i n s N i c 2 1 A d e 2 1 x M e t 2 1

P h e n o t y p i c c l a s s 3 I e t + + + + . . . . A d e + + - - - - ~- --', - - - - N i e + - - + - - + - - + - -

N u m b e r o f i s o l a t e s a 5 3 32 35 b 136 b 18 5 1

a A t o t a l o f 2 3 5 . b P a r e n t a l p h e n o t y p e .

contact of hyphae and increase probabil i ty of fusion. The centre of the produced colony was transferred to the minimal medium with thiamine (5 mg/L) and incubated for 6 d. After a 1-2-d lag complementation and proto- trophic growth were observed (Gold et al. 1982).

The heterokaryot ic mycelium was then inoculated on Petri dishes contain- ing the modified Gold fruit-body-formation-inducing medium (Gold and Cheng 1979) of the following composition (g/L): MgSO4.TH20 0.5, KH2PO4 0.6, K2PO4 0.4, ammonium tar t ra te 0.18, thiamine 10 mg/L, cellulose (What- man) 10, agar 20. Before spreading, the medium was supplemented with 0.1 mM cAMP. After inoculation the dishes were incubated for 5--7 d. The plates were then transferred to the laboratory and turned upside down. Nine to twenty-one days after the inoculation fruit bodies were formed and basidiospores were produced. A 1-4-d yield of basidiospores was washed from the cover and spread onto the complete sorbose medium. After a 4-d incubation the grown colonies were isolated and their nutritional requirements were determined.

Concentrations of nutritional factors in the test minimal medium were as follows (mg/L): methionine 50, nicotinic acid 5, adenine 2.5.

In addition to crosses single mutan t • single mutant , the cross double mutant X single mutan t was performed (Tables II, III) .

Similarly to Alic and Gold (1985), we found that genetic recombination takes place in fruit bodies of the heterokaryons. Whereas in the paper b y Alic and Gold (1985) the ratio among recombination classes was significantly shifted in favour of prototrophic strains, which was explained by contami- nation by prototrophic conidiospores, in our experiments the ratio among recombination classes did not, with a single exception (32 Met+Ade-Nic + : 18 Met-Ade+Nic - in cross Nic21Ade21 • Met21), significantly differ from 1 : 1 as evaluated by means of the 72-test and binomial distribution. Micro- scopic observations showed that conidiospores are not present among spread basidiospores at all or only to a low degree ( < 0 .1%). As the age of the basidiospore population spread was not presented in the paper of Alic and Gold (1985) it is also possible that this difference is caused b y a lower viability of double mutants.

The results of Alie and Gold (1985) indicate tha t P. chrysosporium is probably a homothallic species which is also confirmed b y our finding that the auxotrophic strains Nic21 and Sr21 produce fruit bodies and basidio- spores on the fruit-body-formation-inducing medium supplemented with 0 .001% nicotinic acid or 0 . 0 1 % Na2S203.5H20, respectively.

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180 R. KREJ~I: Vol. 32

As in homothallic species an increased fraction of parental genotypes in the progeny is to be expected, the substantially lower proportion of re- combinants in cross Met21 x Ade21 cannot be considered to indicate a genetic linkage. Nevertheless, the use of multiple mutants in the control of regularity of the crosses makes it possible to avoid this distortion (Fincham and Day 19v J).

R E F E R E N C E S

ALto M., GOLD M.H.: Genetic recombination in the lignin-degrading basidiomyceto Pha;~ero- chaete chrysosporium. Appl.Environ.Microbiol. .~0, 2 7 - 3 0 (1985).

BVRDSALL H.H. Jr. , ESLY~ W.E.: A new Phanerochaete with a chrysosporium imperfeet state. Mycotaxon 1, 123-- 133 (1974).

FINCgA~ J.R.S., DAY P.R.: Fnngal Genetics, 3rd ed. Blackwell Scientific Publications, O x f o r d - Edinburgh 1971.

ERicssoN K.-E., J-OH~SRVD S.C.: Mutants of the white-rot fungus Sporotrichum pulverulentum with increased cellulase and ~.D-glucosidase production. Enzyme Microb.Bioteehnol..5, 425 to 429 (1983).

GOLD M.H., CHE~CG T.M.: Induction of colonial growth and replica plating of the white rot basidiomyceto Phanerochaete chrysosporium. Appl.Environ.Microbiol. 35, 1223--1225 (1978).

GoLI) M.H., CHE~,~G T.M.: Conditions for fruit body formation in the white rot basidiomycete Phanerochaete chrysosporium. Arch.Microbiol. 121, 37--41 (1979).

GoLD M.H., CHENO T.M., MAX'FI~LD M.B.: Isolation and complementation studies of auxotrophic mutants of the lignin-dcgrading basidiomycete Phanerochaete chrysosporium. Appl.Environ. Microbiol. 44, 996--1000 (1982).

GoL~) M.H., CHE~-o T.M., AT.Ic M.: Formation, fusion, and regeneration of protoplasts from wild-type and auxotrophic strains of the white rot basidiomycete Phanerochaete chrysospo- rium. Appl.Environ.Microbiol. 46, 260--263 (1983).

JOH~SRUD S.C., ERIKSSO~r K.-E.: Cross breeding of selected and mutated homokaryotic strains of Phanerochaete chrysosporium K-3: New cellulase deficient strains with increased ability to degrade lignin. Appl.Microbiol.Bioteehnol. 21, 320--327 (1985).

PET~EY T.M., C'~AW~'ORD D.L.: Enhancement of lignin degradation in Streptomyces spp. by protoplast fusion. Appl.Environ.Microbiol. 44, 439--440 (1984).

RAEI)ER U., BRODA P.: Comparsion of the lignin-degrading white rot fungi Phanerochaete chry- sosporium and Sporotrichum pulverulentum at the DNA level. Current Genetics 8, 499--506 (1984).

RAEUgE S.E., TATV~ E.L.: Spontaneous and nitrogen mustard induced nutritional deficiencies in Saceharomyee8 cerevisiae. Arch.Biochem. 22, 331--338 (1949).

S~IDER P.J. , RAPER J.R.: -Nuclear migration in the basidiomycete SchizophyUum commune. Am.J.Bot. 45, 538--546 (1958).