Proc. Nati Acad. Sci. USA Vol. 79, pp. 3641-3645, June 1982 Microbiology Replication and expression of a bacterial-mitochondrial hybrid plasmid in the fungus Podospora anserina (Podospora transformation/hybrid shuttle vector/prokaryotic-eukaryotic gene expression) ULF STAHL, PAUL TUDZYNSKI, ULRICH KUCK, AND KARL ESSER Lehrstuhl fir Allgemeine Botanik, Ruhr-Universitat, D-4630 Bochum 1, Federal Republic of Germany Communicated by Kenneth V. Thimann, February 8, 1982 ABSTRACT Hybrid plasmids consisting of the bacterial plas- of genetic material? (iii) Further, will it be possible to use a pl mid pBR322 and plasmid-like DNA (pl DNA) sequences from the DNA sequence excised from juvenile mtDNA as a vector? This fungus Podospora anserina are not only able to replicate in Esch- would open a possibility for a different form of genetic engi- erichia coli but also in the fungus. This was proved by both bio- neering-one using a eukaryotic replicon of mitochondrial or- physical and biological evidence involving buoyant density pro- igin as a vector. files, DNADNA hybridization, and restriction analysis-all Data presented here show that genetic information from bac- confirming that pl DNA behaves as a true plasmid. During its terial DNA integrated with pl DNA can be expressed in trans- amplification in P. anserina, the hybrid plasmid does not lose its formed Podospora anserina. prokaryotic coding capacity as shown after retransfer and subse- quent cloning in E. coli P. ansermna is able to express both the eukaryotic and the prokaryotic genetic information of the hybrid MATERIAL AND METHODS plasmid because the occurrence ofsenescence and the production Strains. The wild strain s (American Type Culture Collection of .-lactamase could be shown in experiments involving specific 26003) and the double mutant grisealvivax (gr: grey asco- hybrid plasmids. In the same systems, it was possible to demon- spores; viv: aerial hyphae rhythmical) of Podospora anserina strate that a hybrid plasmid containing, instead of p1 DNA, a pl were used as recipients in DNA homologous region of native mtDNA also could function as transformation experiments (life span a true plasmid. This hybrid plasmid contained about 25% of the about 25 and >900 days, respectively). (For details on their genetic information ofpl DNA, which corresponds to about 6% of origin, ontogeny, and genetics, see K. Esser in ref. 9.) Escher- the genetic information of mtDNA. Thus, the data show that hy- ichia coli K-12 SF8 (recB21, recC22, lop-11, tonAl, thr-1, brid plasmids may be used to shuttle genetic information between leu-6, thi-1, lacYl, supE44, r-, m-) served as prokaryotic host. P. anserina and E. coli Hence, through the use ofa mtDNA repli- Plasmids. Single (pSP4, pSP24) or double (pSP17) copies of con, as evidenced by the pl DNA of P. anserina, another pathway pl DNA were integrated in the Sal I site of pBR322 (10). In in genetic engineering is established. pSP24 and pSP17, pl DNA is orientated the same; in pSP4, the orientation is inverted (details are in ref. 6). pKP402 is con- The senescence that occurs regularly in wild strains of the as- structed like pSP4 but contains the Sal I fragment 4 of mtDNA comycete Podospora anserina (1) is caused by an infective agent from P. anserina as a eukaryotic constituent (7). (2) identical with plasmid-like DNA (pl DNA) (3, 4). This DNA Methods. Culture conditions and media, isolation of DNA, species is not found in juvenile mycelia and has the following and the determination of buoyant densities were as described properties: contour length, 0.75 tkm; buoyant density, 1.699 g/ by Stahl et al. (6). Transformation procedures were carried out cm3; and molecular size, 2.4 kilobases (kb). Confirmative ob- as described for P. anserina (4) and for E. coli (6). servations were recently reported by Belcour et al. (5). Restriction enzyme analysis. Restriction enzymes were pur- The pl DNA inserted into the prokaryotic plasmid pBR322 chased from Boehringer (Mannheim, Federal Republic ofGer- (6) has been cloned in Escherichia coli, and the hybrid plasmid many). The digestions were performed as recommended by the pSP17 that was isolated from the bacterium was able to induce supplier. Gel electrophoresis was carried out as described (11). senescence injuvenile cultures ofP. anserina (4). Furthermore, Labeling ofDNA. The pl DNA and pBR322 were labeled in heteroduplex analysis and DNA-DNA hybridization (Southern vitro by nick translation (12) with the kit (radioactive component blotting method) have established that pl DNA is an integral [32P]dCTP) from Amersham Buchler (Braunschweig, Federal part of mtDNA from juvenile mycelia of P. anserina (7, 8). Republic of Germany). Specific activities of about 107 cpm/pug The discovery ofpl DNA ofmitochondrial origin in a eukary- of DNA were obtained. otic organism has raised the following questions. (i) Is the pl Blotting and DNA-DNA hybridization. DNA was separated DNA of P. anserina a true plasmid, having the characteristics in agarose gels, transferred to nitrocellulose strips (Sartorius, attributed to bacterial plasmids-i.e., self-replication and G6ttingen, Federal Republic ofGermany) by the Southern (13) expression? The latter has already been shown for pl DNA by procedure, and hybridized to labeled DNA probes as described induction of senescence in juvenile cultures through transfor- (7). The strips were autoradiographed with Kodak X-Omat AR mation experiments (4). Experimental evidence for pl DNA self- film at -70'C for various times. replication will be given in data confirming that pl DNA con- Determination off3-lactamase. The method used was modi- tains an origin of replication (replicon). (ii) Will it be possible fied from those of O'Callaghan and Morris (14) and Hollenberg to use this naturally occurring pl DNA as a vector for the transfer (15). To 3-4 g of mycelium (wet weight), equal amounts of 100 mM phosphate buffer (pH 7.0), designated buffer P1, and glass The publication costs ofthis article were defrayed in part by page charge beads (diameter, 0.1 mm) were added for disruption (30 sec in payment. This article must therefore be hereby marked "advertise- ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Abbreviations: pl DNA, plasmid-like DNA; kb, kilobase(s). 3641 Downloaded by guest on September 30, 2021 3,542 Microbiology: Stahl et aL Proc. Nati. Acad. Sci. USA 79 (1982) a homogenizer; Braun, Melsungen, Federal Republic of Ger- and (Fig. 1) comprised the following criteria: (i) there were no many). After centrifugation at 10,000 x g for 10 min, the su- homologous sequences of both tester DNAs (pBR322 and pl pernatant (0.1-1.8 ml, variable in (3-lactamase content) was DNA) with nuclear DNA; (ii) as expected, the pl DNA hybrid- made up to 1.9 ml with buffer Pi and mixed with 0. 1 ml ofbuffer ized only with mtDNA from which it originated; and (iii) the Pi containing nitrocephin (5 mg in 10 ml), a chromogenic de- "new" DNA species found after transformation in P. anserina rivative of cephalosporine. (3-Lactamase converts the yellow had homologous sequences with both testers (pBR322 and pl color of this substrate into a red cleavage product that can be DNA)-i.e., the component sequences of pSP17. measured by absorbance at 390 nm. (Nitrocephin was a gift from These results can be considered as strong evidence for the Glaxo Research, Greenford, England.) replication ofthe hybrid plasmid pSP17 in P. anserina because Containment. All transformation experiments were carried the bacterial plasmid pBR322 is not able to amplify in this fun- out under L2/B1 conditions according to the "Richtlinien zum gus. From this it follows that pl DNA must contain the replicon SchutzvorGefahren durch invitro rekombinierte Nukleinsauren" that is able to initiate the replication of the hybrid DNA mol- of the Bundesminister fur Forschung und Technologie. ecule pSP17 in P. anserina. Biological evidence. In order to ascertain whether, after its RESULTS amplification in the eukaryote, the hybrid plasmid pSP17 had In the assessment of the self-replication potential of pl DNA, retained its capacity to replicate and be expressed in a prokary- the cloning ofthe hybrid plasmid pSP17 (consisting ofpBR322 otic system, this plasmid derived from both the wild strain and and two copies of pl DNA) in E. coli is not determinative be- gr/viv was used for transformation ofE. coli. Because the only cause the replication origin of such a hybrid molecule might available marker of pSP17 is the ,B-lactamase gene, ampR, E. stem from the prokaryotic part of this vector. Therefore, we coli cells were screened for ampicillin-resistant transformants. attempted to clone this plasmid in P. anserina, where only the For a further characterization, the DNA of the E. coli clones eukaryotic part may initiate replication. It was established in was isolated and submitted to restriction analysis and Southern early experiments (4) that the prokaryotic part of this vector blotting (Table 1). In addition hybrid plasmid pSP24, which (pBR322) is neither amplified nor expressed when transferred contains only one copy ofpl DNA, was assayed under the same to P. anserina. conditions. During the course of these experiments, the possibility of The data from Table 1 reveal the following facts. (i) As com- expression of the genetic information ofthe prokaryotic part of pared with the control (pBR322), a rather high rate oftransfor- pSP17 in a eukaryotic host could be tested (10). In this context, mation in E. coli was obtained with the hybrid plasmid pSP17. it must be recalled that the plasmid pBR322 carries, in addition This means that the cloning of this plasmid in P. anserina had to the tetracycline-resistance gene whereby the pl DNA is in- not altered its capacity to amplify and to be expressed in the serted, a gene causing resistance to ampicillin and coding for prokaryotic host.