Curr Genet (1991) 19:149-153 Current Genetics �9 Springer-Verlag 1991

Short communication

Transformation of Penicillium roqueforti to phleomycin- and to hygromycin B-resistance Nathalie Durand, Pascale Reymond, and Michel Ftvre

Laboratoire de Biologie Cellulaire Fongique, UMR CNRS 106 Universit6 Lyon I (Brit. 405) 43 Boulevard 11 Novembre 1918, F-69622 Villeurbanne C4dex, France

Received October 17, 1990

Summary. A strain of Penicillium roqueforti was trans- formed to hygromycin B and phleomycin resistance us- ing resistance genes under the control of A. nidulans se- quences. The t ransformat ion efficiency ranged f rom 0.15 to 1 t ransformant per gg D N A per l 0 6 viable protoplasts when t ransformants were selected on medium containing a high antibiotic concentrat ion (7 -10 times the min imum inhibitory concentration). Transformat ion resulted f rom either single copy or tandem integration of the phleo- myein vector while the hygromycin vector was modified during integration. The t ransformed antibiotic-resistant phenotypes were mitotically stable with or without selec- tive pressure.

Key words: Penicillium roqueforti - Transformat ion - Phleomycin Hygromycin

Introduction

Penicillium roqueforti is a fi lamentous fungus used in the dairy industry. Hybridizat ion and breeding have provid- ed opportunit ies for creating new Penicillium strains or improving some of their useful properties (Ann6 1982, Reymond et a1.1986). However, t ransformat ion systems are needed so that a broad array of the genes, encoding technologically impor tant enzymes, can be cloned and experimentally manipulated both in vitro and in vivo.

Several t ransformat ion systems have been described for Penicillium chrysogenum exploited for the product ion of antibiotics. They are based on complementat ion of mutat ions with homologous or heterologous genes (Sanchez et al. 1987; Cantoral et al. 1987), or on domi- nant selectable markers such as the acetamidase gene (Beri and Turner 1987), G418 resistance (Stahl et al. 1987), phleomycin resistance (Kolar et al. 1988), benomyl

Offprint requests to: N. Durand

resistance (Picknett and Saunders 1987), and sulfon- amide resistance (Carramolino et al. 1989). Penicillium species used for food processing have also been trans- formed. A chlorate-resistant mutan t of Penicillium casei- colurn wag t ransformed by the nitrate reductase gene of A. nidulans (Daboussi et al. 1989), and Penicillium nal- giovense was successfully t ransformed with the amdS sys- tem (Geisen and Leistner 1989).

In this report we describe the t ransformat ion of Peni- cillium roqueforti to hygromycin resistance by the vector pAN7-1, which carries the E. coli hygromycin B phos- photransferase gene (Punt et al. 1987), and to phleomycin resistance by the vector pUT771 containing the phleo r gene characterized f rom Tn5 (Collins and Hall 1985).

Materials and methods

Strains and plasmids. A Penicillium roqueforti strain from our labo- ratory culture collection was used in this study. This strain was maintained on PDA and cultured on complete medium (Reymond and F~vre 1986). Plasmid pAN7-1 was constructed by Punt et al. (1987). It contains a E. coli hygromycin B resistance gene (hph) cloned downstream of the glyceraldehyde 3-phosphate dehydroge- nase gene promoter from A. nidulans and in front of a sequence which contains the A. nidulans trpC transcription termination sig- nal. Plasmid pUT771 was a gift from Cayla (France). It contains a phleomycin resistance gene (phleo~), cloned under the control of a fungal promoter, and the trpC terminator from A. nidulans.

Preparation of protoplasts. Penicillium protoplasts used in transformation experiments were prepared according to the proce- dures previously described (Gaugy and Ftvre 1986; Reymond and F~vre 1986). Conidia were inoculated in 100 ml of complete liquid medium in a 250 ml Erlenmeyer flask. The culture were grown overnight at 25 ~ on a giratory shaker at 120 rev/min. The myceli- um was harvested by filtration, washed with 0.7 M NaCI and then resuspended in a filter-sterilized osmotic buffer (0.7 M NaC1 in 0.1 mM phosphate buffer pH 5.6) containing 10 mg/ml Novozym 234 (Novo Industries, Denmark) and 10 mg/ml cellulase Onozuka (Serva, RFA). After 3 h incubation at 30 ~ under moderate agita- tion, protoplasts were separated from cell debris by filtration through glass wool. The suspension was transferred to sterile cen- trifuge tubes, overlaid with an equal volume of NaC1 solution, and centrifuged at 2000 g for 10 min. The pelleted protoplasts were then washed with a sorbitol solution (1 M sorbitol, 50 mM CaC12) be-

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fore finally being resuspended in the sorbitol solution at a final concentration of 5 x 107 protoplasts/ml. This protoplast suspension was used for the transformation procedure_

Transformation protocol. Transformation was performed essen- tially as described by Yelton et al. (1984). Different concentrations of pAN7 or pUT771 in 20 gl TE buffer (10 mM Tris pH 8.0, I mM Na 2 EDTA) were added to 0.2 ml protoplast suspension (5 x t07 protoplasts/ml). After 20 rain incubation at room temperature, 200 gl, 200 gl and then 600 gl of PEG solution (50% PEG 6000 in 10 mM Tris HC1 pH 7.5/50 mM CaCI2) were added and mixed gently. Protoplast suspensions were then kept on ice for 20 rain. A further 1 ml of the PEG solution was added, mixed gently and the suspensions incubated for 5 rain at room temperature. Cells were diluted in 10 ml of the sorbitol solution and centrifuged at 2000 g for 10 rain. Protoplasts were plated on appropritate selective media or transferred into regeneration liquid medium before plating on agar medium.

Isolation and analysis of DNA. Penicillium transformants were grown in liquid cultures in the presence of hygromycin (t-ImB, Sigma, St. Louis), or phleomycin (phleo, Cayla) for 4 days at 25 ~ DNA from freeze-dried mycelium was isolated according to the method described by Raeder and Broda (1985). Restriction enzyme analysis, E. coli transformation, agarose gel electrophoresis and.i Southern blotting were carried out according to standard protocols= Hybridization probes were nick translated and hybridized under stringent conditions (Maniatis et al. 1982).

Results

Table 1. Transformation ofPenicillium roqueforti to hygromycin B- and phleomycin-resistance

Selected Antibiotic Transformation marker concentration frequency (vector) of the selective (number of trans-

medium (gg/ml) formants/gg DNA)

Hygromycin 20 J 0.00 (pAN 7-1) 200 0.44

300 0.32

Phleomycin 45 1.00 (pUT 771) 50 0.88

60 0.15

At these high hygromycin B and phleomycin concen- trations, resistant colonies were respectively visible after 4 and 8 days at 25 ~ Transformation frequencies ob- tained for different levels of selective pressure are sum- marised in Table 1. All the HmB transformants were morphological ly similar. In addition to fast-growing phleomycin transformants , a number of smaller "abort ive colonies" appeared but did not grow when transferred to fresh medium containing phleomycin.

Transformation. The minimum inhibitory concentrat ion (MIC) of the antibiotics used as selection markers was determined by plating spores on nutritive agar medium containing different amount of H m B or of phleo. Growth was not observed on plates containing 20 gg HmB/ml or 10 gg Phleo/ml. Regeneration of protoplasts on osmoti- cally stabilized agar medium was completely inhibited at these concentrations. For protoplas t production, each 100 ml of Penicillium cultures yielded 10 v protoplasts with 48% viability after transfer to non-selective medium. After P E G treatment, protoplas t viability was only 10%. When, following t ransformation, selection of HmB R or Phleo R t ransformants of P. roqueforti was real- ized on agar medium at the M I C of hygromycin or phleomycin, numerous rapid growing colonies appeared after 3 - 4 days. The frequency was about ten per gg of DNA. However, when these colonies were transferred to fresh selective medium, most of them did not maintain the antibiotic-resistant phenotype. This indicates that they were abortive t ransformants as the yield of sponta- neous muta t ion of resistance to each antibiotic was less than 10 7 for hygromycin B and less than 10 s for phleomycin.

In order to prevent selection of abortive transfor- mants, the selection procedure was modified. Following t ransformation, protoplasts were transferred to an os- motically stabilized liquid medium, incubated for 14 h at 25 ~ and then plated on solid media containing 200 and 300 gg/ml of hygromycin or 60 gg/ml of phleomycin. This long regeneration period prior to the transfer on selective media was necessary for the expression of drug resistance. No resistant colonies were recovered when protoplasts were transferred to these selective media im- mediately after t ransformat ion or after short regenera- tion periods (less than 8 h).

Analysis of genomic DNA from the transformants

Hygromycin-resis tant and phleomycin-resistant trans- formants were randomly selected for the analysis of the t ransformant DNAs. Transformants were grown in liquid medium containing hygromycin or phleomycin and total D N A was isolated and analyzed by hybridiza- tion. Undigested and restricted genomic DNAs were elec- trophoresed through a 0.8% agarose gel, transferred to a nitrocellulose membrane and hybridized to [ a2P]-labelled pAN7-1 or pUT771 plasmid DNAs.

As shown in Figs. 1 and 2, D N A from all the transfor- mants showed strong hybridization signals to labelled pAN7-1 or pUT771, whereas the D N A of the recipient strain had no homology with these vectors. In undigested D N A from both types of t ransformants , hybridization signals occured only with high molecular weight DNA, strongly suggesting the absence of au tonomously repli- cating vector molecules and the integration of the trans- forming vectors into the genome. Digestion of hy- gromycin B-t ransformant D N A s was performed with HindIII which cuts once in the vector pAN7-1 (Punt et al. 1987). As shown in Fig. 1, two types of t ransformants were detected. Transformants 1 and 3 were characterized by two hybridization bands as expected for single site integration, indicating that a single copy of the vector was integraded into the genome. D N A hybridizations with t ransformants 2 and 4 yielded a single signal band of 8 kb suggesting rearangements and deletions during the integration events. This was confirmed when transfor- mant DNAs were restricted with EcoRV which does not cut in pAN7A; a single hybridization signal, ranging f rom 5 to 6 kb, was detected for each t ransformant (Fig. 1). As pAN7.1 lacks an EcoRV site, signals higher than 6.5 kb were expected. These results indicate that

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Fig. 1. Soutiaern hybridization analysis of Penicillium roqueforti transformants. Undigested-DNA, EcoRV- and Hind III-digested DNA, run on 0.8% agarose gels, was transferred to nitrocellulose and probed with [32p]-labelled pAN 7-1. Lane WT, wild-type DNA; lanes T1-T4, DNA from transformants. Molecular size markers (kb) were from Hind III-digested lambda DNA. The plasmid size is indicated by the arrow

10

o

A

o _ _

0 0.5 1 1o5 Hygromycin B concentration ( mg/ml

16

12

- o

45

o I 1

o 0;2 0;4 0 .8

B Phleomycin concentration ( mg/ml )

Fig. 3A, B. Growth ofPenicillium reoqueforti wild-type and trans- formants with different levels of hygromycin B (A) and phleomycin (B). Mycelial growth was estimated as the colony diameter (cm) after 3 days at 25~ Each point is the average of five measure- ments. A growth in the presence of hygromycin. Wild-type ([]) and transformants TI (o), T2 (,), T3 (,,), T4 (o). B growth in the presence of phleomycin. Wild-type (n) and transformants S 1 (o), $2 (A), $3 (=), $4 (o)

Fig. 2. Southern hybridization analysis of Penicillium roqueforti transformants. Undigested-DNA, Bam H1- and Sma l-digested DNA, run on 0.8% agarose gels, was transferred to nitrocellulose and probed with [32P]-labelled pUT 771. Lane WT, wild-type DNA; lanes S1-$3, DNA from transformants. Transformant $4 exhibited a hybridization pattern similar to that of $1. Molecular size markers (kb) were from Hind III-digested lambda DNA. The plasmid size is indicated by the arrow

deletion of vector sequences (i.e., disappearance of the Hindl I I site) had occurred during integration and that pAN7.1 was integrated at a single locus. As those hy- bridization bands do not have the same size, we may suspect that integration occurred at different sites in each t ransformant .

A more complex pat tern of hybridization was ob- served between the D N A of phleomycin t ransformants and pUT771 which has a single Srna 1 site and no Barn H1 site and is 6.15 kb in size (Calmels T. personnal communication). After digestion with Srna 1, an intense 6.15 kb band was observed in addition to two bands of different size. This hybridization pat tern is expected if the plasmid integrated in tandem repeats: i.e., a hybridizing band corresponding to the size of the vector and other bands representing vector genome border fragments. The presence of tandem integrated repeats of pUT771 was confirmed by the hybridization pat tern of Barn HI-di - gested t ransformant DNAs. Large D N A fragments (20 kb), i.e., more than twice the size of the vector, hy- bridized to the probe as expected for a tandem repeat.

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Antibiotic resistance and mitotic stability

Transformants exhibited high levels of resistance to the antibiotics (Fig. 3 A, B). For example, some HmB R trans- formants were resistant to 1 500 gg/ml hygromycin B (70 times the MIC) and Phleo R transformants were resistant to 800 gg/ml phleomycin (80 times the MIC). These transformants were stable after several subcultures on non-selective medium using a mycelium plug as an inocu- lum. Mitotic stability was also observed with spores. Two hundred spores from transformant colonies were plated on complete medium without antibiotics, and grown for 3 days at 25 ~ After sporulation, the colonies on CM were replicated on CM supplemented with antibiotics. All plated spores gave rise to resistant colonies showing that the transformants remained stable.

Discussion

We have described two DNA-mediated transformation systems for P. roqueforti based on dominant selectable markers: hygromycin B- and phleomycin-resistance. The hygromycin system has been widely used for transforma- tion of several different filamentous fungi (Punt et al. 1987; Oliver et al. 1987; Mullaney et al. 1983; Cooley etal . 1988; Wnendt etal . 1990). By contrast, the phleomycin system based on the Streptoalloteichus hin- dustanus ble gene (Mattern and Punt 1988; Durand et al. 1988) or the Tn 5-derived ble gene (Calmels, Cayla) have rarely been used. Since both selection systems require the expression of heterologous genes, it is obvious that the production of transformants indicates that the A. nidu- lans expression signals which control these resistance genes are functional in Penicillium.

The transformation frequency of P. roqueforti proto- plasts to hygromycin resistance was comparable with the frequencies obtained for several Aspergillus species (Punt et al. 1987; Mullaney et al. 1988; Wnendt et al. 1990), for Curvularia lunata (Osiewacz and Weber 1989), Fusarium oxysporum (Kistler and Benny 1988), Botryotinia squamosa (Huang et al. 1989) and Glomerella cingulata (Rodriguez and Yoder 1987).

Transformation efficiency to phleomycin resistance was also low but comparable to the results obtained with A. nidulans and the plasmid pAN8-1 (Mattern and Punt 1988; van Engelenburg et al. 1989).

Southern blot analyses revealed that transformation proceeds via integration of the transforming plasmids into the P. roqueforti genome. The patterns of hybridiza- tion observed between the restricted D N A of the trans- formants and pAN7-1 showed that deletion of the trans- forming vector had occurred. This was illustrated by the disappearance of the HindIII restriction site. Rearrange- ments of the vector sequences, and/or the genomic D N A of the recipient cell, have also been shown to occur in other systems (Razanamparany and Begueret 1988; Mohr et al. 1989). Whether these recombinations are produced during integration or following growth on se- lective medium remains to be determined. As we have been using high antibiotic concentrations to select trans-

formants, it is possible that only transformants harboring a very stable and efficient integration of the dominant marker were selected. This integration could be favoured by heterologous rearrangments and deletions. Analysis of phleomycin-resistant transformants showed hy- bridization patterns which are more commonly found in other systems. Plasmids became integrated either as sin- gle copies or as tandem arrays. Both type of transfor- mants showed a high level of resistance to the antibiotics and a very high stability after growth on non-selective media.

Drug-resistance markers are useful for construction of gene transfer systems in genetically poorly character- ized organisms. The data presented here demonstrate that DNA-mediated transformation of P. roqueforti can be used for the stable introduction of specific D N A se- quences. The transformation systems described will also provide a good basis for further studies including the introduction of selectable dominant markers into indus- trial strains by protoplast fusion and the use of P. roque- forti strains for the expression of heterologous genes.

Acknowledgements. The authors wish to thank Dr. P. J. Punt (The Netherlands) and Dr. T. Calmels (Cayla, Toulouse, France) for respectively providing pAN7-1 and pUT771.

References

Ann6 J (1982) Environ J Appl Microbiol Technol 15:41-46 Beri R, Turner G (1987) Curr Genet 11:639-641 Carramolino L, Lozano M, Perez Aranda A, Rubio V, Sanchez F

(1989) Gene 77:31-38 Cantoral JM, Diez B, Barredo JL, Alvarez E, Martin JF (1987)

Biotechnology 5:494-497 Collins CH, Hall RM (1985) Plasmid 14:143-151 Cooley RN, Shaw RK, Franklin FCH, Caten CA (1988) Curr

Genet 13:383-389 Daboussi M J, Djeballi A, Gerlinger C, Blaiseau PL, Bouvier J,

Cassan M, Lebrun MH, Parisol D, Brygoo T (1989) Curr Genet 15:453-456

Durand H, Baron M, Calmels T, Tiraby G (1988) In: Aubert JP, Beguin P, Millet J (eds) Biochemistry and genetics of cellulose degradation. Academic Press, New York, pp 135-151

Engelenburg F van, Smit R, Goosen T, Van den Brock H, Tudzyn- ski P (1989) Appl Microbiol Biotechnol 30:364-370

Gaugy D, Fevre M (1986) Microbios 44:285-293 Geisen R, Leistner L (1989) Curt Genet 15:307-309 Huang D, Bhairi S, Staples RC (1989) Curr Genet 15:411-414 Kistler HC, Benny UK (1988) Curr Genet 13:145-149 Kolar M, Punt P, Van den Hondel CAMJJ, Schwab H (1988) Gene

62:127-134 Maniatis H, Fritsch EF, Sambrook J (1982) Molecular cloning: a

laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York

Mattern JE, Punt PJ (1988) Fungal Genet Newslett 35:25 Mohr G, Wilmanska D, Esser K (1989) Appl Microbiol Biotechnol

32:160-166 Mullaney E J, Punt P J, Van den Hondel CAMJJ (1988) Appl Micro-

biol Biotechnol 28:451-454 Oliver RP, Roberts IN, Harling R, Kenyon L, Punt P J, Dingen-

manse MA, Van den Hondel CAMJJ (1987) Curr Genet 12:231-233

Osiewacz HD, Weber A (1989) Appl Microbiol Biotechno130: 375- 38O

Picknett JM, Saunders G, Ford P, Holt G (1987) Curr Genet 12:449-455

Punt P J, Oliver RP, Dingenmanse MA, Pouwels PH, Van den Hon- del CAMJJ (1987) Gene 40:99-106

Raeder U, Broda P (1985) Lett Appl Microbiol 1:17-20 Razanamparany V, Begueret J (1988) Gene 74:399-409 Reymond P, Fevre M (1986) Enzyme Microbiol Technol 8:41-44 Reymond P, Veau P, Fevre M (1986) Enzyme Microbiol Technol

8:45 -47 Rodriguez RJ, Yoder OC (1987) Gene 54:73-81

153

Sanchez F, Tourino A, Trasiera S, Perez-Avanda A, Rubio V, Penal- va MA (1987) Gene 51:97-102

Stahl U, Leitner E, Esser K (1987) Appl Microbiol Biotechnol 26: 237-24t

Wnendt S, Jacobs M, Stahl U (1990) Curr Genet 17:21-24 Yelton MM, Hamer JE, Timberlake WE (1984) Proc Natl Acad Sci

USA 81:1470-1474

C o m m u n i c a t e d by K. W o l f