PLASMID14, 126-133 (1985)

Isolation and Characterization of an Extrachromosomal Element from Nocardia mediterranei

PAOLA MORETTI, GILBERTO HINTERMANN, AND RALF HUTTER

Mikrobiologisches Insiitut, Eidgeniissische Technische Hachschuie Ziirich, ETH-Zentrum, CH-8092 Ztirich, Switzerland

Received February 8, 1985; revised May 29, 1985

Strain LBG A3 136 of Nocardia medizerranei (ETH Collection) was found to contain a low- copy-number covalently closed circular extrachromosomal element, pMEAlOO, which could only be isolated from mycelium grown on agar plates. pMEAlO0 could not be isolated from the closely related strain ATCC 13685. Hybridization experiments showed that pMEA 100 is present in strain LBG A3 136 in the free as well as in the integrated form whereas in strain ATCC 13685 only an integrated form was detected. Excision and reintegration in strain LBG A3 136 seemed to be site specific. pMEA 100 was found to be self-transmissible, eliciting the lethal zygosis phenotype, and is possibly involved in fertility in N. mediterranei. Q 1985 Academic press, 1~.

Nocardia mediterranei is a nonsporulating actinomycete of industrial interest, producing the ansamycin-type antibiotics rifamycins (Sensi and Thiemann, 1967). A circular link- age map of chromosomal markers was estab- lished, based on segregational data after agar surface matings (Schupp et al., 1975; Schupp and Niiesch, 1979). The mating frequency re- sembled that of Streptomyces coeiicolor A3(2) IF matings (IF is the “initial fertility” of S. coelicolor wild-type A3(2)) (Hopwood et al., 1973). No mutants with altered mating be- havior were yet found, and no extrachromo- somal element, which could possibly be in- volved in fertility, was reported.

Our interest in the development of plasmid cloning vectors for N. mediterranei initiated a more thorough search for plasmids in this spe- cies, using various wild type and mutant strains. We report on the isolation and char- acterization of an extrachromosomal element occurring in certain N. mediterranei strains.

MATERIALS AND METHODS

Strains and plasmids. The actinomycete strains are in Table 1, plasmids are in Ta- ble 2.

Propagation and storage of strains. Nocar- dia and Streptomyces strains were usually

propagated on CM agar plates: yeast extract (Oxoid) 4 g; malt extract (Difco) 10 g; D&U- case 4 g; agar (Bacto agar, Difco) 20 g; distilled water to 1000 ml; adjusted to pH 7.2-7.5 with 2 M NaOH prior to autoclaving (Pridham et al., 195611957). For growth in flasks, LM me- dium was used: sucrose 103 g; yeast extract (Difco) 3 g; peptone (Difco) 5 g; malt extract (Oxoid) 3 g; ~-glucose 10 g; glycine 10 g; MgC12. 6 Hz0 2 g; distilled water to 1000 ml. Strains were stored as 20% glycerol suspen- sions at -20°C.

Isolation of pMEA IO0 from Nocardia med- iterranei. (Modified from Kieser, 1984.) The mycelium scraped from four well-grown CM plates (3 days, 30°C) was resuspended in 5 ml lysozyme solution (10.3% sucrose; 25 mM Tris-HCl, pH 8.0; 25 mM EDTA, pH 8; 10 mg/ml lysozyme) and incubated at 30°C for l-2 h. Then, 3.5 ml lysis solution (0.3 M NaOH; 2% sodium dodecyl sulfate (SDS)) were added and the suspension was thoroughly mixed. After 10 min at room temperature the suspension was incubated at 80°C for a further 10 min and cooled to room temperature. A 5- ml phenol-chloroform mixture (500 g phenol; 500 ml chloroform; 500 mg 8-hydroxyquin- oline) equilibrated with distilled water, was added and the solution was strongly mixed on a Vortex mixer until the appearance was milky

0147-619X/85 $3.00 Copyright 0 1985 by Academic Press. Inc. All rights of reproduction m any form reserved.

126

EXTRACHROMOSOMAL ELEMENT FROM N. mediterranei 127

TABLE 1 STRAINS

Designation Description Source a

Nocardia mediterranei LBG A3 136 T88

ATCC 13685

ATCC 21643 TlOl Tll8 N813

ATPll8 12298 S2800 NB66 W126

Nocardia asteroides N8 N61 KK4

Streptomyces tolypophorus ATCC 21177 IF0 12554

Streptomyces lividans TK21

Streptomyces giaucescens ETH 22794

wild type, see Ghisalba et al., 1984 ETH pdx-2, lys-I, str-2, see Schupp, 1973 T. Schupp, derived from

LBG A3 136 ME83/973 derivative, MnCl#eated wild-type strain (see ATCC

Ghisalba et al., 1984) wild type ATCC pro-l, str-2, am-4 (Schupp and Niiesch, 1979) > pur-13 rifamycin B high producer (Ghisalba and Ntiesch, 1977) T. Schupp, sttrains

derived from ATCC 13685

rifamycin nonproducing mutant low rifamycin B producer rifamycin S producer rifamycin B producer rifamycin W producer

K.P.S. K.P.S. K.P.S.

ATCC IF0

JII

wild type ETH

a ETH: strain collection of the Mikrobiologisches Institut ETH, Zihich, Switzerland. T. Schupp: strains obtained from T. Schupp, Ciba-Geigy AG, Basel, Switzerland. ATCC: American Type Culture Collection, Washington DC. KPS.: strains obtained from K. P. Schaal, Hygiene-Institut der Universitit, Cologne, West Germany. JII: strain obtained from John Innes Institute, Norwich, England. IFO: strain obtained from Institute for Fermentation, Osaka, Japan.

and homogeneous. The phases were separated by centrifugation (10,OOOg for 10 min). A sample of the upper phase (30-80 ~1) was loaded on an agarose gel for preliminary plas- mid detection. The upper phase was trans- ferred to a new tube and the DNA was pre- cipitated with l/ 10 vol of 3 M sodium acetate and 1 vol of isopropanol. The DNA was sed- imented ( lO,OOOg, 10 min) and resuspended in TE buffer (10 mM Tris-HCl, pH 8; 1 mM EDTA). This DNA solution was further pu- rified by cesium chloride-ethidium bromide centrifugation (150,000g for 40 h). For rapid screening for plasmid presence in agarose gels,

mycehum was scraped from about one-fourth of a well-grown CM plate and resuspended in 500 ~1 lysozyme solution. Further treatment was as indicated above.

Isolation of total DNA. The mycelium was grown in WI-ml flasks (with bafhes) contain- ing 50 ml LM medium for 2-4 days at 30°C on a rotary shaker. DNA isolation was ac- cording to Hintermann et al. (198 1).

Hybridizations. Southern transfer, prehy- bridization, hybridization, and autoradiogra- phy were done according to Maniatis et al. ( 1982). Hybridizations were carried out in 50% formamide, 5X SSC (1X SSC: 0.15 M NaCl;

128 MORETTI, HINTERMANN, AND HiiTTER

TABLE 2

PLASMIDS

Designation

pIJ41

pIJ303

pIJ409

pIJ702

pMEA 100

Description”

SLP 1.2 derivative, tsr+, up/r+

pIJ IO I derivative, tsr’

pBR325-pIJ408 hybrid plasmid, blu+, cut+, vph+

pIJlO1 derivative: mel+, tsr+

endogenous ccc DNA of N. mediterranei LBG A3 136

Source b

JII (Thompson et al., 1982)

JII (Kieser et al., 1982)

JII (Hopwood et al., 1984)

JII (Katz et al., 1983)

This work

pMEA101

pMEA103

pBR322-pMEA 100 hybrid plasmid, b/u+

Hybrid plasmid: pBR322 with the 6. l- kb BumHI fragment of pMEA100: bla’

This work

This work

pMEA 104 Hybrid plasmid: pBR322 with the 6.4- kb BumHI fragment of pMEA 100: bla+

This work

pMEAIO5 hybrid plasmid: pBR322 with the 10.5- kb BumHI fragment of pMEA 100: b/a+

This work

’ uph’: neomycin phosphotransferase; blu+: ampicillin p-Iactamase; cut+: chloramphenicol acetyhransferase; meI+: S. antibioticus tyrosinase; tsr+: 23s rRNA pentose methylase (confers resistance to thiostrepton); tyr’: 5’. gluucescens tyrosinase; vph+i viomycin phosphotransferase.

’ JII: John Innes Institute.

0.0 15 M sodium citrate) at 42°C; membranes were washed in 2X SSC; 0.1% SDS at 68°C (three times, 30 min). Nick translation was done using [32P]CTP (3000 Ci/mmol) accord- ing to Maniatis et al. (1982). Dot blots were according to Thomas (1980).

Genetic techniques. Ethidium bromide and acridine orange mutagenesis were done ac- cording to Hiitter et al. ( 198 1) and Suter et al. (1978), respectively. The method of proto- plasting and protoplast regeneration of N. mediterranei was according to T. Schupp and M. Divers (manuscript in preparation). Transformation experiments were done ac- cording to Bibb et al. (1978). Crosses were made and analyzed according to Schupp et al. (1975).

RESULTS

Characterization of ccc DNA from certain Nocardia mediterranei strains. ccc DNA could

be purified from N. mediterranei strain LBG A3 136 in cesium chloride-ethidium bromide gradients. The element detected, pMEA100, had a size of about 23.7 kb. The copy number was estimated to be low, possibly below 0.5 copy of extrachromosomal element per hap- loid genome, based on hybridization experi- ments (see below). ccc DNA of the same size could be isolated from the mutant strain T88, a direct descendant of strain LBG A3 136, but no DNA was detected in two other N. medi- terranei strains (ATCC 13685 and ATCC 27643), either in agarose gels or in cesium chloride-ethidium bromide gradients; the de- tection level was in the range of 0.0 1 copy per haploid genome.

A restriction map of pMEA 100 is shown in Fig. 1. (see Table 3 for additional data). In addition to the sites shown, pMEA100 also has four sites for KpnI, three sites for XhoI, and more than nine sites each for BglI, BstEII,

EXTRACHROMOSOMAL ELEMENT FROM N. mediterrunei 129

pMEAlO0 23,l kb

lo= 65,8 bp

FIG. I. Restriction maps of pMEAlO0 and pMEAIOl. The restriction sites are numbered (see Table 3 for the distances). Only the sites which were exactly mapped are shown. The flag indicates the SphI insertion site and the orientation of pBR322 in pMEA 10 I.

HindII, MluI, PvuII, SalGI, and SmaI. There was no site for EcoRI.

Hybridization ofpMEAlO0 to “‘total” DNA of various Nocardia mediterranei strains. In order to simplify recovery and restriction en- zyme analysis, and in order to avoid hybrid- ization artifacts caused by contamination of pMEA 100 preparations with chromosomal DNA fragments, pMEAlO0 was cloned in Escherichia coli strain ED8767 on pBR322, leading to pMEA101 (Fig. 1). In addition, three of the four BamHI fragments of pMEA100 were separately cloned onto pBR322, leading to pMEA103 (6. I-kb frag- ment), pMEA104 (6.4-kb fragment), and pMEA105 (10.5-kb fragment). (The 0.8-kb BamHI fragment of pMEA 100 was not cloned.)

N. mediterranei strains were analyzed for the presence of DNA sequences homologous to pMEA101. Total DNA was isolated, di- gested with BamHI, subjected to agarose gel electrophoresis, and hybridized to “P-labeled pMEA 10 1 DNA. The results are shown in Fig. 2. Any hybridization observed must be due to

pMEAlO0 sequences in the probe because pBR322 does not hybridize with N. mediter- ranei DNA.

The wild-type strain LBG A3136 and its derivative strain T88 exhibited, in addition to the expected three bands of 10.5, 6.4, and 6.1 kb, two additional bands of 3.5 and 3.3 kb (the 0.8-kb fragment was not visualized in these experiments). Furthermore, the 6.1 -kb BamHI fragment was weaker than expected, suggesting that pMEA100 might, at least in part, exist as an integrated form and that in- tegration occurred within the 6.1-kb BamHI fragment. This was verified by the hybridiza- tion pattern with pMEA 103, pMEA104, and pMEA105 (Fig. 3), where pMEA103, con- taining the 6.1 -kb BamHI fragment, was the only plasmid hybridizing with the 3.5- and the 3.3-kb bands.

TABLE 3

MAP DISTANCES FOR pMEAl00’

Site Restriction No. enzyme

I BumHI 2 BamHI 3 Hind111 4 BgAl 5 BglII 6 SUCI 7 Hind111 8 SphI 9 Clal

10 EcoRV II SphI 12 BamHI 13 BgnI 14 Cld 15 BclI 16 BclI 17 BumHI 18 BgnI 19 sac1 20 CIaI 21 Bell 22 SUCI

23 C/al 24 Bcfi

25/l BamHI

’ Data for Fig. I.

Distance from site 1 (kb)

0 0.8 1.8 2.4 2.82 3.1 3.3 3.85 4.6 4.6 5.8 6.85 7.7 1.85 9.35 9.85

13.2 13.3 13.4 14.8 17.4 19.1 21.2 21.4 23.7

Distance between sites (kb)

0.8 1 0.6 0.42 0.28 0.2 0.55 0.75 0 1.2 1.05 0.85 0.15 1.5 0.5 3.35 0.1 0.1 1.4 2.6 1.7 2.1 0.2 2.3

130 MORETTI, HINTERMANN, AND HOTTER

(A) (6) -

105kb-

Kr I--

-105 kb

-70 kb -6Lkb 761 kb

59kb

,35kb -33 kb

we-30kb

FIG. 2. Hybridization of ‘*P-labeled pMEA 10 I DNA to BumHI digests of N. mediterranei DNA. Running con- ditions were 0.75% agarose for 15 h at 2 V/cm; IO’ cpm 32P-labeled pMEA 101 were used for hybridization. (A) represents the gel after electrophoresis. (The plasmid bands in lanes 1 and 2 of the agarose gel are weak, since low concentrations of DNA were purposely loaded to avoid too strong signals in the hybridization.) For total DNA of N. mediterranei 3.5 rg were loaded in each slot. (B) shows the X-ray film at’ter 3 h exposure. (X) XHindIII, (1) pMEAlO0, (2) pMEA101, (3) LBG A3136 (the same pat- tern was obtained with strain T88), and (4) ATCC 13685 (the same pattern was obtained with strains TlOl, Tll8, N8 13, ATPI 18, 12298, S2800, NB66, and W 126).

The situation in strain ATCC 13685 and its derivatives was found to be more complex (Fig. 2). The bands at 10.5, 6.4, 3.5 and 3.3 kb were in common with strain LBG A3136, but the 6.1 -kb fragment was lacking and extra bands of 7.0, 5.9 and 3.0 kb were seen. Anal- ysis with the three cloned BamHI fragments of pMEA100 (Fig. 3) revealed hybridization of the three extra bands with either the 10.5- kb or the 6.4-kb fragment of pMEA100, while the 6.1 -kb fragment hybridized only to the two fragments of 3.5 and 3.3 kb, presumably rep- resenting the integrated form as in strain LBG A3136.

pMEA 10 1 did not exhibit any hybridization to N. mediterranei strain ATCC 27643, an in- dependent isolate of the species from New Zealand. All Nocardia and Streptomyces strains listed in Table 1 were tested and none of them showed hybridization to pMEA 10 1. No cross hybridization was observed between pMEA 100 and the streptomycete plasmids pIJ41, pIJ303, and pIJ702, and only a very weak hybridization was found with pIJ409.

isolation of pMEAlOO-free derivatives of Nocardia mediterranei LBG A3136 and T88. In order to obtain pMEA 1 00-free derivatives, treatment with ethidium bromide and acridine orange were performed, but even at a dye con- centration leading to less than 0.1% survivors, no cured progeny were obtained. However, pMEA loo-free strains could be obtained with a frequency of about 30% from regenerants of protoplasting experiments. Of 60 colonies an- alyzed 20 protoplast regenerants showed no hybridization with labeled pMEA 10 1 in dot- blot experiments. Four of these were culti- vated further. All of them exhibited some hybridization after regrowth. They were subject to another round of protoplasting. From each parent four regenerants were taken and none of them showed any hybridization even after several rounds of recultivation. They were taken as completely devoid of pMEA 100 DNA.

Conjugational transfer of pA4EAlOO and pock formation. Plating of a mixture of pMEA 100&e and pMEA 1 OO-containing cells on CM agar led to the formation of pocks (Fig. 4), however, only about 20% of the colony forming units produced pocks. Similar pocks have been reported in several cases as being due to plasmid spread from plasmid-carrying to plasmid-free cells (Bibb et al., 1977; Kieser et al., 1982). Mycelium taken from N. medi- terranei pocks was grown on agar selective for

--

- =- -

-- --

Hybrldlsatwns wth fragments of pMEAlO0

--

-- -

-- -- -

FIG. 3. Schematic representation of the hybridization results using BarnHI fragments of pMEA100. (1) pMEAlO5 (105kb fragment), (2) pMEA104 (6.4-kb frag- ment), and (3) pMEA 103 (6. I-kb fragment).

EXTRACHROMOSOMAL ELEMENT FROM N. mediferrunei

FIG. 4. Pocks produced on a pMEA loo-free lawn of N. mediterranei LBG A3 I36 by pMEA IO@containing LBG A3 136 cells.

the originally pMEA loo-free partner strain T88 of the plating mixture. Analysis of this culture yielded DNA preparations with ccc DNA, while mycelium taken from pock-free areas was plasmid-free. This supports the hy- pothesis of plasmid transfer and plasmid spread in pocks of N. mediterranei.

In some cases a ccc DNA preparation in- distinguishable from pMEA100 by BamHI digestion was isolated from mycelium origi- nating from pocks. However, often more than one ccc DNA type was seen in a preparation. Restriction analysis indicated that such prep- arations contained the original element pMEA 100 in variable amounts and, in addi- tion, different deletion derivatives of it. A pre- cise location of single deletions was not pos- sible because of the lack of a transformation system for N. mediterranei and the failure to detect transformation of S. lividans (data not shown).

pMEA 100 was also found to be a fertility factor for the transfer of chromosomal mark- ers. In crosses involving at least one pMEA loo-carrying parent, recombinant fre- quencies of 10e4 to 10m3 with respect to the total cell counts, or low3 to lo-* with respect to the minority parent (strain T88) were ob-

served (Table 4). No recombinants were ob- tained in crosses between two pMEA loo-free strains.

DISCUSSION

In this work, we describe the isolation and characterization of pMEA 100, an extra-

TABLE 4 RECOMBINATION FREQUENCIES BETWEEN N. Mediterranei STRAINS LBG A3 136 AND T88

Recombination frequencies”

cross 1 2

LBG A3136 p+ X T88 p+ 1 o-4 10-Z LBGA3136p+xT88p- 10-3 10-z LBG A3136 p- x T88 p+ lo-’ 10-z LBG A3 136 p- X T88 p- <lo+ 110-n

Note. Strain LBG A3 136 (pdx’, lys+, sfr’) was crossed with strain T88 (p&, lys-, strr). Selection was for pdvf sfr’ or lys+ str’. For both selections we obtained about the same number of recombinants. (p’: pMEAlOO+; p-: pMEA IOO-).

a 1: Recombinant frequency with respect to the total cell counts of both parent strains. 2: Recombinant fre- quency with respect to the minority parent (parent present in lower amount).

132 MORETTI, HINTERMANN, AND HijTTER

chromosomal element of N. mediterranei. pMEA 100 could only be isolated from strain LBG A3136 (ETH collection) and the deriv- ative strain T88, but not from the ATCC 13685 strain.

Hybridization experiments with labeled pMEAlO1, pMEA103, pMEA104, and pM- EA105 indicate that pMEA 100 can exist as free or integrated forms in strains LBG A3 136 and T88, and that the 6. I-kb fragment origi- nates by fusion of the two ends of the inte- grated form of pMEA 100. Several reports exist about plasmids arising from chromosomal ex- cision events (Hopwood et al., 1984; Omer and Cohen, 1984). In the case of pMEA100 we find site-specific excision as reported for other cases (Omer and Cohen, 1984), since the 6. I-kb BamHI fragment gives a discrete band in digests.

The intensity of the 6.1-kb BarnHI fragment relative to the other bands in hybridization experiments with labeled pMEA 101 is pre- sumably directly related to the amount of free element present in the strain; the low intensity of this band indicates that the copy number of the free form of pMEA 100 per haploid ge- nome is below 1 (possibly lower than 0.5). This would also explain why only low amounts of ccc DNA can be isolated from the cultures (see Materials and Methods).

Protoplast regeneration of strain LBG A3 136 (and T88) leads to a very high per- centage of loss of pMEA 100 (about 30%). To- tal DNA of cured derivatives did not exhibit any hybridization to pMEA 100. This suggests that in single cells or cell equivalents pMEA 100 exists either in the integrated or in the free form, and that the cured strains arise from cells which harbored the free element. Loss of other plasmids after protoplast regen- eration has been described earlier (Hopwood, 1981).

The arrangement of pMEA 100 sequences in strain ATCC 13685 and its derivatives is more complex than in strain LBG A3 136. The presence of the two bands at 3.5 and 3.3 kb and the absence of a 6.1 -kb fragment suggest that pMEA 100 is present only in the integrated and not in the free form. Parts of the plasmid sequences might also be integrated at other

sites, as revealed by the bands at 7.0 and 5.9 kb hybridizing to the 10.5-kb BamHI frag- ment, or the 3.0-kb band hybridizing to the 6.4-kb BarnHI fragment of pMEA 100 (Fig. 3).

DNA homologous to pMEA 100 was only found in N. mediterranei LBG A3136 and ATCC 13685 (and their derivatives). Other Streptomyces and Nocardia strains tested did not show any hybridization to pMEA 100, in- cluding N. mediterranei strain ATCC 27643 isolated in New Zealand. Of the few strepto- mycete plasmids tested only pIJ409, isolated from S. lividans after interspecific mating with S. glaucescens (Hopwood et al., 1984), showed very weak hybridization to pMEA100, likely due to unspecific interaction rather than seg- ment specific homology.

The fact that pMEA100 has an extremely low copy number per chromosome suggests that it may not be a replicative element. Pre- liminary experiments also indicated that no replication occurs in S. lividans. Possibly pMEA 100 is an element propagated only as part of the chromosome and not in the free form.

pMEA 100 can be transferred in N. medi- terranei from a pMEAlOO-harboring to a pMEAlOO-free strain. The transfer is accom- panied by the formation of pocks. pMEA100 also seems to function as a fertility factor for the transfer of chromosomal markers and re- combinant formation. In crosses between two pMEA loo-free strains no recombinants were recovered. In crosses with at least one pMEA 1 00-carrying partner a recombinant frequency was obtained similar to that de- scribed by Schupp et al. ( 1975). Many reports exist about fertility plasmids in Streptomyces (Hopwood et al., 1973; Friend et al., 1978; Bibb and Hopwood, 198 1; Kieser et al., 1982; Lydiate, 1984), while for N. asteroides con- jugational fertility is postulated to be plasmid- independent (Kasweck et al., 198 1).

ACKNOWLEDGMENTS

We are grateful to T. Schupp for providing us with N. mediterranei strains, and for the information about re- generation of protoplasts. This work was supported by re- search grants from the Swiss Federal Institute of Tech- nology, Ziirich, and from the “Kommission zur Fijrderung der wissenschaftlichen Forschung” (project 1231).

EXTRACHROMOSOMAL ELEMENT FROM N. mediterranei 133

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