1. business and industrial
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Vol. 57, No. 3
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1991, p. 665-671
0099-2240/91/030665-07$02.00/0
Copyright © 1991, American Society for Microbiology
Construction of a Hybrid Plasmid Capable of Replication in
Amycolatopsis mediterranei
RUP LAL, SUKANYA LAL,t ERWIN GRUND, AND RUDOLF EICHENLAUB*
Lehrstuhl fur GentechnologielMikrobiologie, Fakultat fur Biologie, Universitat Bielefeld, Postfach 8640,
W4800 Bielefeld 1, Federal Republic of Germany
Received 27 August 1990/Accepted 20 December 1990
Several species of bacteria belonging to the order Actinomycetales are widely used for the production of antibiotics.
In this context, "Nocardia mediterranei," recently reclassified as Amycolatopsis mediterranei (16), and A. orientalis
are of special interest as they produce the commercially and
medically important rifamycin and vancomycin antibiotics
(3, 16, 30). In addition, several strains of Amycolatopsis
have been recently reported to metabolize aromatic hydrocarbons (9).
Methods for gene cloning have been developed for several
species of Streptomyces (1, 15, 20, 31). Although A. mediterranei belongs to the same order (Actinomycetales),
recombinant DNA techniques were not available for this
organism. This was mainly due to the lack of any plasmid
suitable for vector development in A. mediterranei; furthermore, standard transformation procedures as used in Streptomyces spp. are not applicable to this organism. So far
conjugation has been the only technique available for introducing DNA into A. mediterranei (29), whereas limited
success has been obtained in transforming A. orientalis with
Streptomyces vectors (21).
Several attempts have been made to develop a suitable
vector system for genetic exploitation of A. mediterranei.
Plasmid pMEA100 was isolated from this bacterium (24).
This plasmid could not be developed into a suitable cloning
vector as it has a low copy number, integrates into the
chromosome (18), and is difficult to isolate from liquid
culture. Subsequently, Schupp and Divers (28) developed a
method for protoplast preparation and regeneration for A.
mediterranei. However, several attempts to transform those
protoplasts with a range of different cloning vectors derived
from Streptomyces such as pIJ61, pIJ702, and pIJ922 were
not successful (28). Thus, further progress in the development of gene cloning techniques in A. mediterranei was
hampered. In this paper we describe the construction of a
hybrid plasmid capable of replicating in A. mediterranei.
This hybrid plasmid was constructed by cloning a 5.1-kb
fragment of pA387, retaining the origin of replication, into
pDM10. We have used electroporation to transform A.
mediterranei and A. orientalis since this technique has been
recently used with success in microorganisms which were
either resistant to normal transformation procedures or did
not previously have any efficient means for genetic transformation (2, 10, 14).
MATERIALS AND METHODS
Bacterial strains and culture conditions. The bacterial
strains and plasmids used in this study are listed in Table 1.
"Amycolatopsis sp." strain DSM 43387, A. mediterranei,
A. orientalis, and A. rugosa were obtained from the German
culture collection (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH [DSM], Braunschweig, Federal
Republic of Germany). Bacteria were usually grown in 148G
medium (28) containing the following, in grams per liter:
glucose, 22; yeast extract, 0.5; beef extract, 4; peptone, 5;
tryptone, 3; glycine, 10; at 28°C on a rotary shaker. For
growth on agar plates either PY agar (Bacto-Peptone [Difco],
5 g; yeast extract [Difco], 3 g; and agar, 15 g, per liter [28])
or GYM agar (glucose, 4 g; yeast extract, 4 g; malt extract,
10 g; CaCo3, 2 g; and agar, 12 g, per liter) was used. Strains
of "Amycolatopsis sp." harboring plasmid pRL1 were
grown in liquiil medium under selective pressure of neomycin (100 jig/ml).
Escherichia coli SF8 was grown in TBY medium containing the following, per liter: tryptone, 10 g; yeast extract, 5 g;
and NaCl, 5 g (pH 7.2). E. coli strains harboring plasmid
pDM10 were grown in liquid medium under appropriate
*
Corresponding author.
t Present address: Sri Venkateswara College, Dhaula Kuan, New
Delhi-110021, India.
t Present address: Department of Zoology, University of Delhi,
Delhi-110007, India.
665
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A new plasmid, pA387, has been isolated from "Amycolatopsis sp." (DSM 43387). This plasmid could be
isolated from liquid culture as well as mycelium from agar plates by a modified procedure. Plasmid pA387 is
about 29.6 kb and can be cured at low frequency by protoplasting and ethidium bromide and heat treatment.
Hybridization experiments showed that this plasmid is present in free form and does not integrate into the
chromosome. A hybrid plasmid was constructed by cloning a 5.1-kb fragment of pA387 into the.Escherichia
coli vector pDM10. This hybrid plasmid, termed pRL1, could be transformed into Amycolatopsis mediterranei
and A. orientalis by electroporation. A transformation frequency of 2.2 x 103 transformants per ,ug of DNA
at 12.5 kV/cm and a pulse duration of 10.8 ms was obtained in A. mediterranei, whereas 1.1 x 105
transformants per ,ug of DNA were obtained at a field strength of 7.5 kV/cm and a pulse duration of 7.6 ms
in A. orientalis. Plasmid pRL1 is the first hybrid plasmid which could be used successfully for the
transformation of A. mediterranei. The plasmid has a rather high copy number, is genetically stable, and can
be easily reisolated from A. mediterranei. Plasmid pRL1 will be useful for further construction of a shuttle
vector for E. coli and A. mediterranei and becomes the basis for the development of gene cloning techniques in
Amycolatopsis spp.
666
LAL ET AL.
APPL. ENVIRON. MICROBIOL.
TABLE 1. Bacterial strains and plasmids used in this study
Species
Strain or
plasmid
Characteristic(s)a
Source or reference
"Amycolatopsis sp."
43387
40773
40040
43194
TK 64
Wild type
Wild type
Wild type
Wild type
pro-2 str-6
hsdR hsdM recBC lop]] ligsuPer thr-l
tonAl leu-6 supE44 lacYl thi-J
29.6 kb
5.8 kb, Tsrr
pACYC184, with Tsrr insert from pIJ702
5.1 kb, Kmr Neor Gnr
10.4 kb, Kmr Neor
DSM
DSM
DSM
DSM
John Innes Institute, Norwich, United Kingdom
R. Davies
A. mediterranei
A. orientalis
A. rugosa
S. lividans
E. coli
SF8
pA387
pIJ702
pSLE41
pDM10
pRL1
Kmr,
Neor, and Tsrr, Conferring resistance to kanamycin, neomycin, and thiostrepton, respectively.
selective conditions, using 50 jig of neomycin or 10 ,ug of
gentamicin per ml.
Plasmid isolation and DNA manipulations. (i) Large-scale
DNA isolation. Large-scale preparations of pA387 from
"Amycolatopsis sp." strain DSM 43387 was performed by
the following procedure. A 1-liter culture in 148G medium
containing 10 g of glycine per liter was grown to stationary
phase, and mycelium was collected by centrifugation and
washed twice with 10% glycerol. Finally, the cells were
resuspended in 54 ml of lysozyme buffer (25 mM Tris
hydrochloride [pH 8], 25 mM EDTA, 300 mM sucrose) and
6 ml of lysozyme mix (lysozyme, 10 mg/ml, and pancreatic
RNase, 50 Rg/ml, in lysozyme buffer) was added. The cell
suspension was incubated at 37°C for 1 h and divided into six
10-ml aliquots. Further purification of DNA was by the
method of Kieser (13). Plasmid DNA was precipitated by
adding 12 ml of isopropanol, and the DNA pellet was finally
dissolved in TES buffer (100 mM Tris hydrochloride, 50 mM
NaCl, 5 mM EDTA [pH 7.5]) followed by further purification
on an ethidium bromide-CsCl gradient (19). Plasmid isolation
from A. mediterranei and A. orientalis was by the method
described for "Amycolatopsis sp." strain DSM 43387 except
that DNA shearing was done at 70°C for 30 min.
(ii) Small-scale DNA isolation. Plasmid pA387 could also be
isolated from mycelium collected from a 5-cm2 area of agar
plates. This method was useful for the rapid screening of
transformants for plasmid DNA. Mycelium from PY agar
plates was collected by centrifugation, washed two to three
times with lysozyme buffer (50 mM glucose, 25 mM Tris
hydrochloride [pH 8.0], 10 mM EDTA), and finally suspended in 100 ml of lysozyme buffer containing lysozyme (10
mg/ml) and pancreatic RNase (50 ,ug/ml). Further steps in
the purification of plasmid DNA were done by the method of
Hopwood et al. (11). The DNA pellet was finally dissolved in
50 ,ul of TE buffer (10 mM Tris hydrochloride 1 mM EDTA
[pH 8.0]). Samples (10 RI) were loaded on a 0.8% agarose gel
for the detection of plasmid DNA.
The boiling method was used for rapid small-scale isolation of plasmid DNA from E. coli (32). For large-scale
preparations, plasmid DNA was isolated by the alkaline lysis
method and purified by ethidium bromide-CsCl gradient
centrifugation (19). Plasmid DNA obtained in this way was
digested with different restriction endonucleases. Whenever
necessary, double digestions were carried out with combinations of endonucleases having single or multiple recognition sites. The digests were analyzed by horizontal agarose
gel electrophoresis. The sizes of restriction fragments were
obtained by comparing their mobility with A DNA fragments
generated by digestion with different restriction enzymes
(HindIII or HindIII-EcoRI or PstI).
Plasmid curing. Plasmid curing in "Amycolatopsis sp."
strain DSM 43387 was carried out by three methods: protoplasting and regeneration, growth at elevated temperature,
and ethidium bromide treatment. For the preparation of
protoplasts and regeneration, the method of Schupp and
Divers (28) was used.
For plasmid curing by growth at elevated temperature,
cultures were first grown at 37°C for 1 day and then shifted
to 40°C and incubated at that temperature for 4 days.
Appropriate dilutions of the culture were plated on GYM
plates, and single colonies were scored after 2 to 3 days.
Plasmid curing by ethidium bromide treatment was carried
out as described by Crameri et al. (5). Appropriate dilutions
of cells were grown on GYM containing 6 ,uM ethidium
bromide (at this concentration only 1% of the colonies
survived). Single colonies appeared after 3 to 4 days.
Colony hybridization. About 100 colonies from a curing
experiment were transferred to a Zetabind nylon filter membrane (Cuno, Inc., Meriden, Conn.) placed on top of GYM
agar plates and grown for 2 to 3 days at 28°C. Zetabind
membrane with fully grown colonies was placed over three
layers of Whatman filter papers (no. 541) soaked with
lysozyme solution (10 mg/ml) and incubated for 2 h at 37°C.
After drying, the Zetabind nylon filter membrane was processed for colony hybridization and color reaction (nonradioactive DNA labeling and detection kit; Boehringer, Mannheim, Federal Republic of Germany).
Southern blot hybridization. Total DNA from "Amycolatopsis sp." strain DSM 43387 was digested with BclI,
subjected to agarose gel electrophoresis, and subsequently
transferred to Zetabind nylon membrane by using Southern
blotting. Plasmid pA387 labeled by nick translation with
digoxigenin-11-UTP was used as a probe. Zetabind nylon
membrane was incubated in the prehybridization mixture at
68°C for 1 h and then in hybridization mixture overnight.
Hybridization was visualized by using a nonradioactive
DNA labeling and detection kit (Boehringer).
Cloning of fragments of plasmid pA387 in E. coli with vector
PDM10. For cloning fragments of pA387, vector pDM10 was
used (22). This vector has the advantage that it has two
antibiotic resistance genes against neomycin or kanamycin
and gentamicin. Plasmid pA387 was subjected to partial
digestion with BclI and ligated to pDM10 digested with
BglII. Digestion with restriction endonucleases, alkaline
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a
This study
John Innes Institute
Wohlleben and Piihler (33)
Meletzus and Eichenlaub (22)
This study
VOL. 57, 1991
RESULTS
Characterization of plasmid pA387. During a screening of
several species of Amycolatopsis for the presence of indigenous plasmids we could only detect plasmids in "Amycolatopsis sp." strain DSM 43387 and A. rugosa. The plasmid
from A. rugosa was found unsuitable for analysis because of
its large size (47 kb). Thus, plasmid pA387, which is smaller,
was selected. A modified procedure for plasmid isolation by
667
""KpnI,5600
22400 KpnI
pA387
29.6Kb
pA-rep
\\uI,1160~tu1100
Kpn I12700
XbaI. 13600
FIG. 1. Restriction endonuclease cleavage map of plasmid
pA387. pA-rep designates the region carrying the information for
autonomous replication in "Amycolatopsis sp." strain DSM 43387.
ethidium bromide-CsCl gradient centrifugation yielded sufficient DNA to establish a restriction map. The plasmid
occurs in low copy number (approximately 10 copies per
cell). The copy number was estimated by comparison of the
amount of plasmid DNA obtained by standard procedure
with the number of CFU and size of the plasmid. By single
and double digests of pA387 with different restriction enzymes and their comparison with A DNA digested with
HindlIl or HindIII-EcoRI, a size of approximately 29.6 kb
was obtained. Plasmid pA387 has single restriction sites for
BglII, XbaI, XhoI, StuI, and NruI. Three sites for KpnI
exist, generating fragments of 7.1, 11.7, and 10.8 kb. Digestion by BcII yields more than 20 fragments which are difficult
to estimate exactly. There are no recognition sites for
EcoRI, BamHI, and HindIII. The physical map of plasmid
pA387 is presented in Fig. 1.
Plasmid curing in "Amycolatopsis sp." strain DSM 43387
was done to obtain a recipient for transformation and because we were interested to know the role of this plasmid in
the degradation of aromatic hydrocarbons. For this purpose
we succeeded in eliminating plasmid pA387 from Amycolatopsis sp. strain DSM 43387 by using three different
methods (protoplasting and regeneration, ethidium bromide
treatment, and heat treatment). Of 10 clones which were
initially selected after colony hybridization, only 7 were
actually cured from the plasmid, as detected by Southern
blot hybridization (data not shown). Plasmid curing occurred
at a frequency of 1 to 4% and heat treatment appeared to be
slightly more effective. In Southern blot hybridization with
BclI-digested total DNA from cured and wild-type strains
and digoxigenin-11-UTP-labeled pA387 as a probe, we never
found any indication for the integration of pA387 into the
chromosome. No phenotypic differences were observed
between the parent strain and the cured derivatives. When
plasmid preparations were examined by agarose gel electrophoresis, in addition to the expected band corresponding to
plasmid pA387, two additional bands of faster migrating
DNA were observed which represent deletion derivatives of
pA387, as shown by hybridization experiments.
Cloning of restriction fragments of plasmid pA387 into E.
coli. Plasmid pA387 appears to be cryptic. We were not able
to detect any phenotypic marker depending on the presence
of this plasmid. We have attempted several times to insert
the thiostrepton resistance gene obtained from pIJ702 or
pSLE41 into this plasmid and to transform "Amycolatopsis
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phosphatase treatment, ligation, and transformation of
CaCl2-treated cells were by the methods of Maniatis et al.
(19). A total of 150 kanamycin-resistant and gentamicinsensitive clones were selected at random and analyzed by
Eckhardt gel electrophoresis (7) for recombinant plasmids.
Protoplast formation and transformation. Protoplasts of A.
mediterranei and A. orientalis were obtained from cultures
grown at 28°C in 148G medium (50 ml) supplemented with 10
g of glycine per liter by the method described by Schupp and
Divers (28). The protoplasts were centrifuged at 3,000 rpm
for 15 min and resuspended in 5 ml of R2L buffer. Protoplasts were then counted by a hemacytometer and centrifuged, and the pellet was resuspended in an appropriate
volume of R2L buffer to obtain approximately 1010 protoplasts per ml.
Protoplasts prepared in this way were transformed with
plasmid DNA by the method described by Hopwood et al.
(12). For each transformation 50 ,ul of protoplasts (1010 cells
per ml) in R2L buffer and 5 ,ul of plasmid DNA (0.1 ,ug/,ul)
were gently mixed in an Eppendorf tube. Immediately after
this, 200 ,ul of 25% polyethylene glycol (PEG) 1000 was
added and gently mixed. Transformed protoplasts were
plated on PYM (PY containing 73.2 g of mannitol per liter)
plates. After about 16 h, when small colonies started to
appear, the plates were overlaid with 2.5 ml of soft agar
containing 750 ,ug of neomycin per ml for A. orientalis and
500 ,ug/ml for A. mediterranei. Neomycin-resistant colonies
appeared after 4 to 5 days. Plasmid pRL1 was isolated from
neomycin-resistant clones and shown to be identical to the
original plasmid by restriction endonucleases analysis.
Transformation of A. mediterranei and A. orientalis by
electroporation. Cultures (100 ml) of A. mediterranei and A.
orientalis were grown to mid-exponential phase in 148G
medium containing 10 g of glycine per liter. Cells were
harvested by centrifugation at 6,000 x g and washed three
times with distilled water. Finally, the cells were resuspended in 5 ml of distilled water and kept on ice before use.
Transformation by electroporation was carried out with a
Gene Pulser apparatus connected to a Pulse Controller
(Bio-Rad Laboratories, Richmond, Calif.). Cell suspension,
50 ,ul, was transferred to a precooled Eppendorf tube and
mixed with 1 to 2 ,ul of pRL1 DNA (0.1 ,ug/,ul). The mixture
was immediately poured into a chilled electroporation cuvette (2 mm; Bio-Rad) and exposed to a single pulse of
varying field strength (5 to 12.5 kV/cm) and pulse duration
(2.2 to 15 ms). Cells were immediately plated on PY agar
plates and allowed to recover for 4 h at 30°C. Thereafter, 2.5
ml of top layer of soft agar containing 500 p.g of neomycin
per ml for A. mediterranei or 750 ,ug/ml for A. orientalis was
added. Transformants were scored after 4 to 5 days. The
neomycin resistance of presumptive transformants was confirmed by growing them in liquid medium containing 100 p.g
of neomycin per ml. The presence of plasmids was confirmed by isolating the plasmid from either a culture grown
on PY agar plates containing 50 ,ug of neomycin per ml or a
liquid culture in 148G containing 100 jig of neomycin per ml.
PLASMID REPLICATION IN A. MEDITERRANEI
668
APPL. ENVIRON. MICROBIOL.
LAL ET AL.
4165.B9l]
sp." by the procedure of protoplasting and PEG treatment,
as applicable with Streptomyces lividans (12); however, this
was not successful. Also, several modifications of the
method, such as the use of different PEG concentrations,
different types of PEG, culture age, variation in cell number,
and heat treatment of protoplasts before transformation to
overcome possible restriction barriers, did not yield any
transformants. Thus, a gene library of pA387 was constructed in E. coli by cloning fragments of plasmid pA387
into pDM10. This vector has its origin from pBR322 and
carries the TnS kanamycin-neomycin resistance gene and
gentamicin resistance of Tn1696 (Fig. 2) (22). For this
purpose a partial digest of pA387 with BclI was ligated with
vector pDM10 linearized by digestion with BglII. Integration
of a DNA fragment into the vector inactivates the gentamicin
resistance gene. Eight clones with recombinant plasmids
representing the entire pA387 plasmid in overlapping fragments were selected. Tentative restriction maps of these
plasmids were generated by standard single and double
restriction enzyme digestions. The clones were found to
contain recombinant plasmids having pA387 inserts of 5.1 to
20.2 kb (Fig. 3). Most of the hybrid plasmids with larger
inserts were unstable in E. coli as they showed deletions
when the cultures were grown for several generations.
Transformation of A. mediterranei by electroporation. Since
standard procedures yielded no successful transformation,
we tested whether electroporation was applicable in Amycolatopsis spp. We could not transform "Amycolatopsis sp."
strain DSM 43387 with any of the hybrid plasmids isolated
from E. coli. However, it was possible to transform A.
mediterranei with the hybrid plasmid pRL1. Initially only 10
transformants per jig of DNA could be obtained with pRL1
isolated from E. coli. Further transformation experiments
were carried out with plasmid DNA isolated from A. mediterranei. The effects of electric field strength and pulse
duration on transformation efficiency were also examined. In
A. mediterranei the highest number of transformants were
obtained when cells were pulsed at 12.5 kV/cm for 10.8 ms,
whereas in A. orientalis lower field strength (7.5 kV/cm) and
shorter pulse duration (7.6 ms) yielded the highest number of
transformants (Fig. 4). However, in A. mediterranei and A.
orientalis transformation by electroporation was distinctly
more effective than that by protoplasting (Table 2).
20.2
FIG. 3. Bcll restriction fragments of pA387 generated by limited
digestion cloned into pDM10. In the center, pA387 with four unique
restriction recognition sites is included for location of the fragments
in relation to the map of pA387.
Plasmid pRL1 prepared from A. mediterranei, A. orientalis, or E. coli was used in electroporation experiments. The
results of these experiments are shown in Table 2. Transformation efficiencies dropped 102- and 105-fold in A. mediterranei and A. orientalis, respectively, when plasmid DNA
was derived from E. coli SF8, although this strain is modification deficient (hsdM).
Analysis of plasmid DNA isolated from transformants of
A. mediterranei and A. orientalis revealed that in all cases
the plasmids were identical to pRL1 as isolated from E. coli
(data not presented). This was further confirmed by Southern blot hybridization (data not shown).
Restriction map of plasmid pRL1. Plasmid pRL1 consists
of a 5.1-kb fragment carrying the origin of replication of
pA387 cloned into the BglII site of pDM10. The 5.1-kb
fragment of pA387 has one restriction site each for XbaI and
KpnI and two restriction sites for BclI. A restriction map of
pRL1 is shown in Fig. 5. The recombinant plasmid contains
the neomycin-kanamycin resistance gene derived from TnS.
Although A. mediterranei has a low-level resistance against
neomycin and grows at a concentration of up to 20 ,ug/ml,
only plasmid-containing strains were able to grow in liquid
medium containing neomycin up to 150 Fg/ml. The plasmid
is present in high copy number (approximately 90 copies per
cell), does not integrate into the chromosome, and can be
isolated with ease for rapid screening of plasmid DNA. In
addition, pRL1 carries two unique restriction sites (HindlIl
and BamHI) which can be used for cloning.
DISCUSSION
In general, occurrence of indigenous plasmids in different
species of Amycolatopsis is not very common. To our
knowledge, plasmids have been reported only for A. orientalis (26) and A. mediterranei (24). Plasmid pMEA100 from
A. mediterranei is not ideal for developing into a cloning
vector as it is present in low copy number, integrates into the
chromosome (18), and thus gives poor yields in plasmid
preparations. Several attempts in related genera such as
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2760, Bam HI
FIG. 2. Restriction endonuclease cleavage map of vector pDM10
(22) Gn and Km resistance genes for gentamicin and kanamycin. ori,
Replication origin of pBR322.
PLASMID REPLICATION IN A. MEDITERRANEI
VOL. 57, 1991
10
669
1000 Transformants/ug DNA
a.
1=
D, 1=
,01
0,
=
I/
D
2
6
4
8
10
Pulse length
14
12
msec
16
18
20
1000 Transformants/ug DNA
1000
Ib .
100
10
10
0,1
0,01 I
0
20
14
16
18
12
msec
FIG. 4. Effect of electric field strength and pulse length on transformation efficiency in A. mediterranei (a) and A. orientalis (b). Electric
field strength of 5 (0), 7.5 (+), 10 (x), and 12.5 (O) kV/cm was generated by directing 1-, 1.5-, 2-, and 2.5-kV electric discharge from a 25-,uF
capacitor, respectively. Different pulse lengths were obtained by directing electric discharge through a pulse controller. Mean values of three
experiments are given.
6
4
2
8
10
Pulse length
members of the family Pseudonocardiaceae, including species of Saccharopolyspora, to use pIJ702 or pWOR120 as a
vector were unsuccessful because of rapid segregation of the
vector under nonselective conditions (8, 21, 28, 34). Our
search for plasmids in different strains of Amycolatopsis was
successful only in "Amycolatopsis sp." strain DSM 43387,
which was found to harbor a plasmid pA387 of 29.6 kb, and
A. rugosa, which had a plasmid of about 47 kb. Although it
HindIII,1
BclI,325
I
9200,K;
pBR-ori
TABLE 2. Transformation efficiency of pRL1 DNA isolated from
different host strains
Transformation
method and strain
pRL1
10.4kb
Transformation efficiency (transformants/,ug of
DNA) with pRLl DNA isolated from:
A. mediterranei
A. orientalis
E. coli
20
>1
>1
7200,E
Protoplasting
A. mediterranei
A. orientalis
20
1.4 x 103
Electroporation
A. mediterranei
A. orientalis
2.2 x 103
1.1 X 105
1.0
X
103
2.0 x 103
1.2 x 105
10
>1
5600, BclI
FIG. 5. Physical map of pRL1. Km, Kanamycin resistance gene;
pBR-ori, origin of replication of vector pBR322; pA-rep, replicon
region of Amycolatopsis plasmid pA387.
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in
t'i
A
U,UU1
670
APPL. ENVIRON. MICROBIOL.
LAL ET AL.
The bacterial source of the DNA to be introduced appears
to be important for successful electroporation of A. mediterranei and A. orientalis. Plasmid DNA extracted from A.
mediterranei or A. orientalis transformed each strain equally
well. However, DNA isolated from E. coli could only be
occasionally introduced into these strains (Table 2). Thus,
"Amycolatopsis sp." strain DSM 43387, A. mediterranei,
and A. orientalis appear to have efficient restriction systems,
which may explain the low transformation rates. This is in
accordance with the observations of MacNeil (17) and Neesen and Volckaert (25), which show that several streptomycetes restrict methylated DNA.
The development of a vector system for A. mediterranei
and related species is a prerequisite for genetic manipulation
of these organisms. Since some strains of Amycolatopsis
produce antibiotics and dispose of the terminal pathways for
the degradation of aromatic hydrocarbons (9), genetic engineering of these properties seems worthwhile. In this context the development of a hybrid plasmid, pRL1, capable of
replicating in A. mediterranei and E. coli and its efficient
transformation into the actinomycete is significant. Plasmid
pRL1 represents a prototype shuttle vector between E. coli
and A. mediterranei. This plasmid already carries two
unique restriction sites (HindlIl and BamHI) useful for
cloning and can thus be improved to become a more suitable
cloning vector. One of the restriction sites (BamHI) can be
used to introduce a second antibiotic resistance marker. In
addition, this plasmid is genetically stable, can be isolated
easily, and is present in high copy number and does not
integrate into the chromosome. Several combinations of
parameters are being tested to improve the transformation
frequency in A. mediterranei, and the extent of the host
range of pRL1 is being investigated. However, this plasmid
could not be introduced into Streptomyces lividans, indicating its specificity to organisms closely related to the genus
Amycolatopsis.
ACKNOWLEDGMENTS
We thank D. Meletzus for providing vector pDM10 and for helpful
discussions. We are also indebted to E. M. Zellerman for technical
assistance.
This work was supported by grant 0319366A from the Bundesminister fur Forschung und Technologie of the Federal Republic of
Germany. Thanks are also due to the Alexander von Humboldt
Foundation for providing a fellowship to Rup Lal.
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2. Allen, S. P., and H. P. Blaschek. 1988. Electroporation-induced
transformation of intact cells of Clostridium perfringens. Appl.
Environ. Microbiol. 54:2322-2324.
3. Barna, J. C. J., and D. H. Williams. 1984. The structure and
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not possible to isolate these plasmids with the minilysis
method from liquid cultures as applicable to Streptomyces
spp. (12), it was possible to isolate pA387 with ease from
mycelium taken from agar plates.
Plasmid pA387 could not be cured easily, and plasmid
curing frequency by protoplasting, growing at elevated temperature, and treating with ethidium bromide was only in the
range of 1 to 4%. Thus, plasmid pA387 is quite stable since
these methods have been reported to give curing efficiencies
of up to 30% in A. mediterranei (24) and Streptomyces spp.
(5). "Amycolatopsis sp." strain DSM 43387, like other
actinomycetes, is filamentous and difficulties are encountered during the selection for plasmid-cured derivatives due
to the appearance of mixed clones. However, we were able
to cure and select clones which were plasmid-free and there
was no need for a second round of curing.
Transformation procedures by protoplasting and PEG
treatment as described for Streptomyces spp. (4, 21, 31) and
electroporation did not yield any transformants with recombinant plasmids in the original host of pA387 "Amycolatopsis sp." strain DSM 43387, but we were able to obtain up to
10 transformants per p,g of DNA by electroporation in A.
mediterranei with the hybrid plasmid pRL1. In subsequent
transformations, using electroporation at high electric field
strength with variable pulse length and pRL1 DNA isolated
from A. mediterranei, it was possible to raise the transformation frequency up to 2.2 x 103 transformants per ,ug of
DNA. Our results with transformation by protoplasting and
PEG treatment yielded an extremely low level of transformation in A. mediterranei. This excluded the possibility of
using this method for genetic manipulation unless improved
further. Electroporation was nearly 103 times more efficient
as compared with protoplasting and PEG treatment (Table
2). Electroporation has, in fact, proved valuable for the
study of bacteria, for which DNA transfer methods are
nonexistent (23), unreliable (27), or less efficient (6).
As compared with A. mediterranei, A. orientalis was easy
to transform with pRL1 DNA. Transformation efficiency of
1.4 x 103 and 1.1 x 105 transformants per ,ug of DNA were
obtained with protoplasting and electroporation, respectively (Table 2). Our results with transformation efficiency
by protoplasting are low as compared with those obtained by
Matsushima et al. (21). They used plasmid pIJ702, which is
smaller in size than pRL1.
was
VOL. 57, 1991
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PLASMID REPLICATION IN A. MEDITERRANEI