JOURNAL OF BACTERIOLOGY, JUlY 1985, p. 180-185 Vol. 163, No.

1
0021-9193/85/070180-06$02.00/0
Copyright © 1985, American Society for Microbiology

Efficient Plasmid Transformation of Streptomyces ambofaciens and

Streptomyces fradiae Protoplasts
PATTI MATSUSHIMA AND RICHARD H. BALTZ*
Department of Molecular Genetics, Lilly Research Laboratories, Indianapolis, Indiana 46285
Received 8 March 1985/Accepted 22 April 1985

A procedure for efficient transformation of Streptomyces ambofaciens and Streptomyces fradiae protoplasts
with plasmid DNA was developed. Transformation frequencies with S. fradiae protoplasts were strongly
influenced by the temperatures for cell growth, protoplast formation, and protoplast regeneration. Transfor-
mation frequencies for both species were also influenced by the culture age before protoplast formation, the
source and concentration of polyethylene glycol, the transformation-inducing agent, the concentration of
protoplasts used in the transformation procedure, and the number of protoplasts added to regeneration plates.
Transformation frequencies were substantially higher for both species when calf thymus DNA and protamine
sulfate were added to the transformation mix. With S. fradiae, transformation frequencies were much lower
with plasmid DNA prepared from other species than with the same plasmids prepared from S. fradiae,
suggesting that S. fradiae expresses restriction and modification. With the modified transformation procedures
using DNA prepared from homologous hosts, S. ambofaciens and S. fradiae are now transformed routinely at
frequencies of 106 to 107 transformants per ,ug of plasmid DNA.

Polyethylene glycol (PEG)-induced plasmid transforma- grown overnight in tryptic soy (TS) broth containing
tion of Streptomyces protoplasts (13, 40) has allowed rapid thiostrepton (5 ,ug/ml) obtained from E. R. Squibb & Sons,
development of efficient gene cloning in several Streptomy- Inc., Princeton, N.J., and then homogenized. Homogenized
ces spp., particularly in Streptomyces lividans (10-12, 15-17, mycelia (5 ml) were added to a sterile 50-ml Sorvall tube and
20-32, 35, 37-39). However, the transformation procedures centrifuged at 1,100 x g for 10 min. The supernatant was
developed primarily for S. lividans (13, 40) have not given decanted, and the pellet was suspended in 5 ml of a solution
high enough transformation frequencies to permit efficient containing 50 mM glucose, 10 mM EDTA, 25 mM Tris (pH
gene cloning in Streptomyces ambofaciens and Streptomy- 8.0), and lysozyme (5 mg/ml) obtained from Calbiochem-
ces fradiae, the producers of the macrolide antibiotics spira- Behring, La Jolla, Calif. After the pellet was suspended, the
mycin (33) and tylosin (3, 7, 8, 36), respectively. S. mixture was placed in an ice bath for 30 min. A portion (10
ambofaciens is a potentially useful host for gene cloning ml) of a solution containing 0.2 M NaOH and 1% sodium
since it is relatively nonrestricting, unlike most Streptomy- dodecyl sulfate was then added. (Both the lysozyme and the
ces spp. (18). S. fradiae is an attractive host for gene cloning alkaline sodium dodecyl sulfate solutions should be prepared
since many mutants blocked in tylosin biosynthesis have just before use.) The contents of the tube were mixed gently
been isolated which are suitable for homospecific and until clearing was complete. The tube was placed on ice for
heterospecific complementation (7) and since several poten- 5 min, and then 7.5 ml of a solution containing 3.0 M sodium
tial practical applications of cloning for antibiotic yield acetate (pH 4.8) was added and mixed by gentle inversion of
improvement and for formation of novel or hybrid antibiotic the tube. The mixture was further incubated on ice for 1.0 to
structures have been identified (3). 1.5 h and centrifuged for 10 min at 10,000 x g. The clear
We therefore have examined many parameters which supernatant was transferred to a sterile Sorvall tube, and the
influence the efficiency of plasmid transformation of S. DNA was precipitated by adding 2 volumes of chilled
ambofaciens and S. fradiae and report here our results, ethanol and storing the mixture at -20°C for 2 h. The DNA
which have led to approximately 1,000-fold improved trans- was pelleted by centrifugation at 22,500 x g for 30 min and
formation efficiencies over those obtained by the S. lividans then resuspended in 0.5 ml of TE buffer (10 mM Tris [pH 8.0]
procedure (13, 40). Transformation efficiencies of 2 x 106 to and 1.0 mM EDTA). The DNA was transferred to a sterile
5 x 106 transformants per ,ug of DNA in S. fradiae and 0.5 x 1.5-ml Eppendorf tube, and 50 ALI of 3 M sodium acetate (pH
107 to 4.0 x 107 transformants per ,ug of DNA in S. 8.0) was added and mixed. Cold ethanol (1 ml) was added,
ambofaciens can now be obtained by our modified proce- and the tube was placed on dry ice for about 10 min and then
dures. A preliminary report of part of this work was pre- centrifuged at 15,600 x g for 5 min. The DNA was resus-
sented elsewhere (6). pended in 0.5 ml of TE buffer, and the precipitation proce-
MATERIALS AND METHODS
dure was repeated twice. The DNA was washed with 70%
ethanol and then resuspended in 100 RIl of TE buffer. The
Bacteria. S. fradiae Ml (2) and S. ambofaciens ATCC plasmid DNA was purified from chromosomal DNA on a 1%
15154 were used in this study. agarose gel. The plasmid DNA was recovered from the gel
Isolation of plasmid DNA. Plasmids pFJ105 (34) and pIJ702 by using a modification of the procedure of Dretzen et al.
(26), both of which carry the gene for thiostrepton resist- (19) developed by Paul Rostek, Eli Lilly and Co. (personal
ance, were prepared by a procedure modified from that of communication). After electrophoresis the gel was stained
Birnboim and Doly (14). Cultures containing plasmid were with ethidium bromide (5 Rxg/ml) in electrophoresis buffer for
15 min. During observation under UV light, horizontal slits
*
Corresponding author. were made in front of and behind the plasmid band. Strips of
180
VOL. 163, 1985 EFFICIENT PLASMID TRANSFORMATION 181

Whatman DE81 DEAE-cellulose paper cut to a height agar. The plates were further incubated at the same tempera-
slightly greater than the thickness of the gel and a width tures. Thiostrepton-resistant S. ambofaciens transformants
slightly greater than the slit width were inserted into the slits. were counted after 3 to 4 days of incubation, and
The paper strip behind the plasmid band was used to prevent thiostrepton-resistant S. fradiae transformants were counted
contamination by DNA of higher molecular weight. The gel after 7 to 10 days of incubation.
was squeezed firmly against the papers to close the slits and In some cases, additional salt was added to the DNA
moistened with electrophoresis buffer. Electrophoresis was before transformation. Sodium acetate was added to a 0.3 M
resumed until the DNA had entered the paper strips, which final concentration in the DNA solution for S. ambofaciens
was verified by observation with UV illumination. To re- transformations, and sodium chloride was added to a 0.5 M
cover the DNA from the DEAE paper, the strips were final concentration for S. fradiae transformations. Unless
placed in a sterile polypropylene screw cap tube. The paper stated otherwise in the text or figure legends, transforma-
was washed with 5 ml of 10 mM Tris hydrochloride (pH tions were performed without the addition of sodium acetate
8)-10.0 mM KCI and drained thoroughly. Then 5 ml of 10 or sodium chloride to the DNA.
mM Tris hydrochloride (pH 8)-1.0 M NaCl was added to the Protoplast regeneration. The efficiency of protoplast re-
paper, and the mixture was swirled in a Vortex mixer until generation was determined by dividing the number of colo-
the paper was dissolved. The paper was removed by passing nies derived from protoplasts by twice the number of colo-
the mixture through a syringe containing a small amount of nies obtained from sonicated mycelia before protoplast
siliconized glass wool and into a 5-in (12.7-cm) Pasteur formation as described previously (2, 5). (The number of
pipette packed loosely with a very small amount of silicon- colonies obtained from sonicated mycelia is doubled since
ized glass wool. The eluate was collected, diluted with an about 50% of the potential CFUs are ruptured in the sonica-
equal volume of water, and precipitated with 2 volumes of tion process to obtain single cells [2].)
ethanol. The precipitation procedure was repeated twice, RESULTS AND DISCUSSION
and the DNA was washed with 70% ethanol. The DNA was
resuspended in TE buffer, and its concentration was deter- Preliminary studies. Initial attempts to transform S.
mined by absorbance at 260 nm. fradiae Ml with plasmid pFJ105 by the procedure of Bibb et
Transformation of protoplasts. S. fradiae cells were grown al. (13) yielded about one transformant per microgram of
at 29°C in TS broth plus 0.4% glycine (2, 5) to 500 to 1,000 plasmid DNA . The transformation frequency was increased
Klett units. S. ambofaciens cells were grown in TS broth about 103-fold when transformations were carried out with
plus 0.4% glycine to 1,500 to 2,500 Klett units at 29 to 37°C plasmid DNA prepared from S. fradiae transformants. This
(5). The cells were homogenized, sonicated, and washed as suggested that S. fradiae expresses restriction and modifica-
described previously (2, 5). The mycelia were resuspended tion and that restriction severely inhibits transformations
by shaking in an equal volume of P medium (2) containing with unmodified DNA. This is consistent with previous
lysozyme (1 mg/ml) and placed in an ice bath for 1 h. The observations which indicate that S. fradiae restricts the
resulting protoplasts were washed two times with P medium, plaque formation of several broad-host-range Streptomyces
resuspended in an equal volume of P medium, and then bacteriophage (18).
chilled for at least 15 min. The protoplast suspension was A frequency of 1,000 transformants per ,ug of DNA was
warmed to room temperature before transformation. For S. not adequate to allow efficient homologous gene cloning in S.
fradiae and S. ambofaciens, transformations were carried fradiae. Further, the extremely low frequencies of transfor-
out at room temperature as follows. Calf thymus DNA (0.8 mation with plasmid DNA prepared from other species
jg; Calbiochem) was added from a stock solution (0.25 clearly excluded the possibility of heterologous gene cloning
mg/ml) to the bottom of a sterile 1.5-ml Eppendorf micro test to produce hybrid antibiotic structures (3). The transforma-
tube. Protamine sulfate (1.5 jig; Calbiochem) was added to tion frequencies obtained with S. ambofaciens, a relatively
the calf thymus DNA from a 0.1% stock solution in P nonrestricting species (18) that produces protoplasts that
medium, mixed, and incubated for 1 min at 23°C. In a readily regenerate viable colonies (5), by the transformation
separate tube, plasmid DNA in TE buffer was mixed with P method of Bibb et al. (13) yielded only about 104 transform-
medium to a final volume of 10 ,ul; this was then added to the ants per ,ug of plasmid DNA (34).
calf thymus-protamine sulfate mixture. Protoplasts were Since the procedure of Bibb et al. (13) did not yield
diluted threefold in P medium, and 200 ,ul of the diluted satisfactory levels of transformation with S. fradiae and S.
protoplasts was added to the mixture. For S. fradiae trans- ambofaciens, we initiated studies to improve the transforma-
formations, 900 jil of filter-sterilized 55% PEG 1000 (usually tion efficiencies with these two species with DNA prepared
from Fluka Chemical Corp., Hauppauge, N.Y. or Sigma from homologous hosts. Preliminary studies indicated that
Chemical Co., St. Louis, Mo.) in P medium was added to the the efficiency of transformation of S. fradiae was dramati-
mixture. For S. ambofaciens transformations, 500 ,ul of 55% cally influenced by the temperature for cell growth before
PEG 1000 in P medium was added. The contents of the tubes protoplast formation and to a lesser extent by the tempera-
were mixed gently and incubated at room temperature for 1 ture for protoplast formation. The best results were obtained
min. For S. ambofaciens transformations, an additional 400 when cells grown at 29°C were converted to protoplasts by
,ul of P medium was added to bring the final volume to 1 ml. lysozyme treatment at 4 to 15°C and when the resulting
For both S. fradiae and S. ambofaciens transformations, the protoplasts were incubated at 29°C on modified R2 agar for
tubes were briefly swirled in a Vortex mixer. Dilutions in P cell regeneration after transformation. When the cell growth
medium were made quickly, and the transformed protoplasts temperature was increased to 34 and 37°C, the transforma-
were plated rapidly on modified R2 agar plates in soft agar tion frequencies decreased about 10- and 100-fold, respec-
overlays (5). S. fradiae protoplasts were incubated at 29°C, tively. The transformation frequencies were less influenced
and S. ambofaciens protoplasts were incubated at 34WC for by the temperature for regeneration, but gradually decreased
16 to 24 h; then the plates were overlaid with a second R2 with increasing temperature between 29 and 37°C. Previous
soft agar overlay (5) containing thiostrepton to give a final studies had shown that the temperatures for cell growth and
concentration of 25 ,ug/ml after equilibration with the bottom protoplast regeneration can dramatically influence the ef-
182 MATSUSHIMA AND BALTZ J. BACTERIOL.

ficiency of cell regeneration from protoplasts of S. fradiae (5) 1100 o

and that higher temperatures were deleterious. Thus, the
reduced transformation frequencies observed with proto- - 108 10-o
0
plasts prepared from S. fradiae cells grown at relatively high ~~~~~-
'1
temperatures (34 to 37°C) probably result from inefficient cc ol
10 10
regeneration of transformed protoplasts.
The transformation efficiency of S. ambofaciens proto- X
co 107
plasts was insensitive to variations in the temperatures for o

cell growth and protoplast regeneration in the 29-to-37°C c10

Cu

range. S. ambofaciens protoplasts prepared and regenerated

over this range of temperatures yielded very similar re- Cu
co
generation efficiencies in earlier studies (5). L- A20
b 1

Effects of protoplast concentration and stabilizing buffers. E

In the S. lividans procedures (13, 40), protoplasts were o. io3 I a
concentrated about 20-fold before transformation. We found
that concentration of S. fradiae and S. ambofaciens lv
0 1.0 2.0 0 1.0 2.0 3.0
protoplasts tenfold inhibited transformation by about three- 103 Klett Units
to tenfold, whereas dilution of protoplasts about two- to FIG. 2. Effects of cell growth phase before protoplast formation
threefold resulted in two- to threefold increases in transfor- on transformation. S. ambofaciens (panel a) and S. fradiae (panel b)
mation frequencies. Also, the number of protoplasts plated cells were grown in TS broth to various cell densities, and
on modified R2 plates after transformation influenced the protoplasts were prepared. Protoplasts were transformed to
final frequency of transformants. Maximum transformation thiostrepton resistance with plasmid pFJ105 at 7 ng per assay.
frequencies were obtained when about 104 protoplasts were Viable protoplasts per transformation assay (A) were determined by
added per plate. About 10-fold lower frequencies were diluting protoplasts in P medium (untreated with PEG) and plating
on modified R2 agar.
obtained when about 3 x 105 to 1 x 106 protoplasts were
added per plate (Fig. 1).
The most recent modified transformation procedure for S. higher frequencies of transformants than were obtained
lividans uses L medium during the lysozyme treatment to when L medium and T medium were used during these
prepare protoplasts and T medium during the transformation steps. The use of combinations of either P medium during
(40). The original procedure for S. lividans used P medium lysozyme treatment and T medium during transformation or
for both steps (13). We found in transformations of S. L medium during lysozyme treatment and P medium during
ambofaciens and S. fradiae with plasmid pFJ105 that the use transformation gave results equivalent to those obtained
of P medium for both the lysozyme treatment and the when using P medium during both steps. Since there was no
transformation steps resulted in about two- to threefold clear advantage to using either L medium or T medium, but
a disadvantage to using both, we routinely used P medium
for both the lysozyme treatment and the transformation
steps.
105 Effects of cell growth phase on transformation. Figure 2a
shows that with S. ambofaciens, the efficiency of protoplast
regeneration decreased from about 80% to less than 30% of
potential CFUs when cells were grown from about 500 to
2,500 Klett units, but the number of viable protoplasts
increased about fourfold and the number of transformants
increased about three- to fourfold over this range. The ratio
of transformants to viable protoplasts present in the trans-
formation mix before the addition of PEG was relatively
cn
constant over this cell density range (ca. 10-3). Thus, the
efficiency of transformation does not seem to correlate
mS
E 104 directly with the efficiency of protoplast regeneration but
0 may be more influenced by the absolute number of viable
protoplasts in the assay. In any event, protoplasts prepared
I.-
1o from cells grown to 1,500 to 2,500 Klett units gave the best
results, in this case about 2 x 104 transformants per 7 ng of
DNA or about 3 x 106 transformants per ,ug of DNA.
The efficiency of protoplast regeneration of S. fradiae
protoplasts decreased only moderately (from 80 to 50%
maximum regeneration) as the cell density before protoplast
formation was increased from 1,500 to 3,000 Klett units (Fig.
2b). The number of viable protoplasts increased about 10-
103 fold, but the number of transformants decreased by 10-fold
104 105 lol over this range. Thus, with S. fradiae, the efficiency of
Protoplasts/Plate transformation is not totally dependent upon the number of
FIG. 1. Effects of protoplast concentration on transformation. viable protoplasts per assay or the efficiency of protoplast
Protoplasts of S. ambofaciens (A\) and S. fradiae (0) were trans- regeneration. It appears that the growth phase of the cells
formed to thiostrepton resistance with plasmid pFJ105 at 7 ng per has a large influence on the efficiency of transformation. The
assay. best results are obtained when cells are grown to about 500
VOL. 163, 1985 EFFICIENT PLASMID TRANSFORMATION 183

heterologous DNA. It has been shown that the addition of

protamine sulfate to bacteriophage transfection or transfor-
mation mixes with Escherichia coli spheroplasts can cause
increased frequencies of transfection and transformation (1,
4, 9). Addition of low levels of protamine sulfate to the
Streptomyces transformation mix caused substantial en-
hancement of transformation frequencies with S. ambo-
faciens and S. fradiae protoplasts; the enhancement was
even greater when calf thymus DNA was also added to the
CO) assay (Fig. 4). The maximum transformation frequencies
were obtained when protamine sulfate was added at 1.5 ,ug
CO) per assay and calf thymus DNA was added at 0.8 ,ug per
E assay (Fig. 4). In the absence of protamine sulfate and calf
0
thymus DNA, transformation frequencies were much lower
with both species (data not shown). The order of addition of
protamine sulfate, calf thymus DNA, transforming DNA,
i03
CO104 2 0 4 0 6 protoplasts, and PEG was also important. The highest trans-
formation frequencies were obtained when the order of
addition was as follows: calf thymus DNA, protamine sul-
fate, plasmid DNA, protoplasts, and then PEG. The trans-
formation frequency was 10-fold lower when plasmid DNA
was added first, followed by the addition of protamine
sulfate and then calf thymus DNA, and it was fourfold lower
1 02 when the addition of plasmid DNA was followed by the
10 20 30 40 50 60 addition of calf thymus DNA and then protamine sulfate. It
PEG (%) therefore appears that binding of protamine sulfate to the
carrier calf thymus DNA is important for efficient trans-
FIG. 3. Effects of source and concentration of PEG on transfor- formations.
mation. S. ambofaciens protoplasts were transformed with plasmid Enhancement of transformation in Streptomyces spp. by
pFJ105 at 7 ng per assay. The Fluka PEG 1000 (0) was from lot the addition of calf thymus DNA might be due to competitive
number 207858-182 and the Koch-Light PEG 1000 (A) was obtained
from David Hopwood. PEG (%) indicates the concentration (wt/vol) protection against endonucleolytic cleavage of plasmid DNA
of PEG before being mixed with protoplasts. before or during uptake by protoplasts. The enhancement of
transformation by protamine sulfate might also be due to the
inhibition of endonuclease (1) or to the formation of DNA-
to 1,000 Klett units. This corresponds to the late- protamine complexes which might penetrate the protoplasts
exponential-to-early-transition phase of growth before sta- more effectively (9). However, we have no data to support
tionary phase (2). The efficiency of transformation per either of these mechanisms.
protoplast was about 0.5 x 10-3 to 5 x 10-3 with protoplasts Effects of DNA concentration and salt concentration on
prepared from cells in this growth phase and about 106 transformation. It was shown in a transformation procedure
transformants per ,ug of DNA. for S. lividans that the addition of certain inorganic salts
Effects of PEG on transformation. PEG induces plasmid caused reproducibly small increases in transformation fre-
transformation of Streptomyces protoplasts. In the S. quencies (T. Kieser, personal communication). We have
lividans procedures (13, 40), 20% PEG induced maximum shown that the addition of 0.3 M sodium acetate to the DNA
transformation. In our initial studies on transformation of S.
fradiae and S. ambofaciens, we found that several different
preparations of PEG induced transformation most efficiently
when higher concentrations were used. A particular lot of
PEG (obtained from Fluka Chemical Corp.) gave highest
transformation frequencies when a 55% solution was added
to the transformation mix (Fig. 3). At 20% PEG, 100-fold
fewer transformants were obtained. However, Koch-Light
PEG obtained from D. Hopwood, John Innes Institute, and
used in the S. lividans transformation studies (40) gave
maximum transformation when a 20% solution was added to
W0,
>. 10'
C1

E
the transformation mix (Fig. 3). Higher concentrations gave w0
a marked inhibition (100-fold at 55% PEG). 103
In other experiments, we noted that most sources of PEG
(including several lots from Sigma Chemical Co.) gave a b
maximum transformation when a 55% solution was added to
the transformation mix. However, some lots reproducibly 0 1 2 3 4 0 0.5 1 2 3 4
gave up to 10-fold lower maximum transformation frequen- Protamine Sulfate (pg/assay) Calf Thymus DNA (ug/assay)
cies than those reported in Fig. 3. Therefore, the source and FIG. 4. Effects of protamine sulfate and calf thymus DNA on
concentration of PEG are very important for efficient trans- transformation. S. ambofaciens (A) and S. fradiae (0) protoplasts
formation, and it is useful to test several sources of PEG and were transformed with plasmid pFJ105 at 7 ng per assay. In panel a,
to use only those that give high frequencies of transformants. calf thymus DNA was kept constant at 0.8 ,ug per assay. In panel b,
Enhancement of transformation by protamine sulfate and protamine sulfate was kept constant at 1.5 p.g per assay.
184 MATSUSHIMA AND BALTZ J. BACTERIOL.

(40), and we obtained equally high transformation frequen-

cies with both procedures. Since our procedure uses about
50-fold fewer protoplasts, it yields a higher frequency of
transformed protoplasts. At 10 ng of DNA, which often gives
maximum transformation efficiency in our system, the ratio
of DNA molecules to viable protoplasts is usually about 50
to 150, and the efficiency of transformation per viable
protoplast ranges between 0.1 and 1.0% for both S.
ambofaciens and S. fradiae. It is not known whether higher
frequencies of protoplast transformation are technically fea-
sible with PEG treatment.
The procedure described here should facilitate homolo-
E gous gene cloning in both S. ambofaciens and S. fradiae and
d possibly heterologous gene cloning in S. ambofaciens, a
ccd
co
strain that is relatively nonrestricting (18). Mutants of S.
fradiae defective in several restriction systems have been
isolated recently (P. Matsushima and R. H. Baltz, Abstr.
Annu. Meet. Am. Soc. Microbiol. 1984, H128, p. 112) and
should be useful for heterologous gene cloning in this impor-
1$- 10 101
0~~~~
0 0 tant species as well.
ACKNOWLEDGMENTS
1 o3
We thank J. Fayerman and D. A. Hopwood for providing plasmid
vectors, D. A. Hopwood for providing a sample of Koch-Light PEG,
1 10 100 1 10 100 T. Kieser for communicating unpublished information on transforma-
DNA Conc. (ng) tion in S. lividans, P. Rosteck for advice on isolating plasmid DNA
from gels, C. L. Hershberger and E. T. Seno for comments on the
FIG. 5. Effects of DNA concentration and salt concentration on manuscript, and C. A. Alexander for typing the manuscript.
transformation. S. ambofaciens protoplasts were transformed with
plasmid pFJ105 (panel a) and pIJ702 (panel b). Symbols (panels a LITERATURE CITED
and b): 0, control transformation; A, sodium acetate added to 0.3 M
in the DNA solution before transformation. S. fradiae protoplasts 1. Baltz, R. H. 1971. Infectious DNA of bacteriophage T4. J. Mol.
were transformed with plasmids pFJ105 (panel c) and pIJ702 (panel Biol. 62:425-437.
d). Symbols (panels c and d): 0, control transformation; A, sodium 2. Baltz, R. H. 1978. Genetic recombination of Streptomyces
chloride added to 0.5 M in the DNA solution before transformation. fradiae by protoplast fusion and cell regeneration. J. Gen.
Microbiol. 107:93-102.
3. Baltz, R. H. 1982. Genetics and biochemistry of tylosin produc-
solution caused increased transformation of S. ambofaciens tion: a model for genetic engineering in antibiotic-producing
with plasmids pFJ1OS and pIJ702 at DNA concentrations of Streptomyces. p. 431-444. In A. Hollaender (ed.), Genetic
10 ng or below (Fig. Sa and b). The enhancement of engineering of microorganisms for chemicals. Plenum Publish-
transformation observed with pFJ105 was due primarily to a ing Corp., New York.
change in the relationship between the number of transform- 4. Baltz, R. H., and J. W. Drake. 1972. Bacteriophage T4 trans-
ants and the DNA concentration. When sodium acetate was formation: an assay for mutations induced in vitro. Virology
added, the number of transformants increased proportion- 49:462-474.
5. Baltz, R. H., and P. Matsushima. 1981. Protoplast fusion in
ately with the square of DNA concentration (i.e., slope of 2 Streptomyces: conditions for efficient genetic recombination
on a log-log plot) rather than with the first power of DNA and cell regeneration. J. Gen. Microbiol. 127:137-146.
concentration as was obtained in the control lacking sodium 6. Baltz, R. H., and P. Matsushima. 1983. Advances in protoplast
acetate (Fig. 5a). A slope of two suggests that a bimolecular fusion and transformation in Streptomyces. Exper. Suppl.
interaction is stimulated by salt and that more efficient 46:143-148.
transformation is obtained from two DNA molecules than 7. Baltz, R. H., and E. T. Seno. 1981. Properties of Streptomyces
from a single molecule. Although this unusual relationship is fradiae mutants blocked in biosynthesis of the macrolide anti-
highly reproducible with pFJ105, it was not observed with biotic tylosin. Antimicrob. Agents Chemother. 20:214-225.
pIJ702 (Fig. 5b). In this case, increased salt concentration 8. Baltz, R. H., E. T. Seno, J. Stonesifer, and G. M. Wild. 1983.
Biosynthesis of the macrolide antibiotic tylosin: a preferred
simply increased the frequency of transformants obtained at pathway from tylactone to tylosin. J. Antibiot. 36:131-141.
all DNA concentrations up to about 10 ng, but had no 9. Benzinger, R. 1978. Transfection of Enterobacteriaceae and its
apparent effect on the slope. applications. Microbiol. Rev. 42:194-236.
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chloride to the DNA solution increased the efficiency of ments in Streptomyces cloning, p. 53-82. In M. Inouye (ed.),
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S. ambofaciens and using the procedure of Thompson et al. tion of plasmid DNA into Streptomyces at high frequency.
VOL. 163, 1985 EFFICIENT PLASMID TRANSFORMATION 185

Nature (London) 274:398-400. extracellular-agarase gene from Streptomyces coelicolor A3(2)

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