Gene. 116 (1992) 43-49
© 1992 Elsevier Science Publishers B.V. All rights reserved. 0378-1119/92/$05.00
43
GENE 06489
Plasmid cloning vectors for the conjugal transfer of DNA from Escherichia
coli to Streptomyces spp.
(Recombinant DNA; phage 0C3 I; gene disruption; shuttle vectors; cosmids; P1 vector; mobilization; gene transplacement;
SCP2*)
M. Bierman, R. Logan, K. O'Brien, E.T. Seno, R. Nagaraja R a o and B.E. Schoner
Lilly Research Laboratories. A Division of Eli Lilly and Company, Indianapolis, IN 46285.0424 (USA)
Received by C.R. Hutchinson: 12 No~rember 1991; Revised/Accepted: 6 February 19~2; Received at publishers: !1 March 1992
SUMMARY
We have constructed cloning vectors for the conjugal transfer of DNA from Escherichia coil to Streptomyces spp. All
vectors contain the 760-bp oriT fragment from the lncP plasmid, RK2. Transfer functions need to be supplied in trans by
the E. coil donor strain. We have incorporated into these vectors selectable antibiotic-resistance markers (Am R, Th R, Sp R)
that function in Streptomyces spp. and other features that should allow for: (i) integration via homologous recombination
between cloned DNA and the Streptomyces spp. chromosome, (ii) autonomous replication, or (iii) site-specific integration
at the bacteriophage 0C31 attachment site. Shuttle cosmids for constructing genomic libraries and bacteriophage P 1 cloning
vector capable of accepting approx. 10.0-kb fragments are also described. A simple mating procedure has been developed
for the conjugal transfer of these vectors from E. coil to Streptomyces spp. that involves plating of the donor strain and either
germinated spores or mycelial fragments of the recipient strain. We have shown that several of these vectors can be
introduced into Streptomycesfradiae, a strain that is notoriously difficult to transform by PEG-mediated protoplast transformation.
INTRODUCTION
Streptomycetes are known for their ability to synthesize
commercially useful secondary metabolites with a variety
of biological activities (Deshpande et al., 1988; Hopwood,
1989). The macrolide antibiotics tylosin (produced by
Correspondence ta: Dr. B.E. Schoner, Lilly Research Laboratories. Division of Eli Lilly and Company, Lilly Corp. Ctr., Indianapolis, IN 462850424 (USA) Tel. (317) 276-5323; Fax (317) 276-9086.
Abbreviations: Am, apramycin; Ap. ampiciilin; AS 1, streptomycete medium; arts, bacterial attachment site for phage ~C31: Exolll, exonuclease III of E. coil; int¢c'~t, gene specifying site-specific integration function from bacteriophage cpC31; kb, kilobase(s) or 1000bp; Km,
kanamycin; loxP. locus where phage P1 site-specific recombinase acts;
MCS, multiple cloning site(s); ori, origin of DNA replication: PEG, poly-
S.fradiae) and spiramycin (produced by Streptomyces
ambofaciens) are two examples. We have been interested in
the genetics of tylosin and spiramycin biosynthesis, and for
that purpose have cloned the tylosin and spiramycin biosynthetic gene clusters from S.fradiae (Baltz and Seno,
1988; Fishman et al., 1987) and S. ambofaciens (Richard-
ethylene glycol: Pollk° Klenow (large) fragment of E. coil DNA polymerase I; n resistance/resistant: R2, streptomycete regeneration medium:
reppU¢. E. coil ori from pUC plasmid; rep pll. bacteriophage PI lytic ori;
rep pip, bacteriophage P I plasmid ori; rep p! 5A E. coil ori from pACYC 184
plasmid; rep scp2", streptomycete ori from SCP2* plasmid; rep t~, temperature-sensitive ori from Streptomyces ghanaensis plasmid pSW344E; S,,
Streptomyces; Sa.. Saccharopolyspora; Sp. spectinomycin; Th, thiostrepton; TS. tryptic soy; [ ], denotes plasmid-carrier state: ::, novel joint
(fusion or insertion).
44
son et al., 1990), respectively. This work was carried out
with available vectors that were introduced into Streptomyces spp. by PEG-mediated protoplast transformation.
This procedure was not very satisfactory for S. fradiae even
with non-restricting mutants (Matsushima and Baltz, 1985;
Matsushima et al., 1987) because of low transformation
frequencies with plasmid DNA isolated from E. coil, and
because of the time and labor required to prepare protoplasts (Rao et ai., 1987). Inter-generic transfer of plasmids
from E. coii to Streptomyces spp. was demonstrated initially
by Mazodier et al. (1989), who incorporated oriT from
RK2 into a replicating plasmid pPM803, and subsequently
by Smokvina et al. (1990), who constructed pSAM2-based
integrative vectors that incorporated the oriT region and
could be conjugally transferred from E. coii to Streptomyces
spp.
To determine if conjugal transfer of plasmids would facilitate genetic analysis of S. fradiae, we constructed different kinds of conjugal vectors, including those which can
integrate by homologous recombination into the genome,
those which can replicate autonomously, and those which
can integrate site-specifically using the bacteriophage ~C31
integration functions. Mating conditions are described for
the introduction of these vectors into S. fradiae and other
Streptomyces spp.
TABLE I
Plasmids constructs
Plasmid
Size
kb
Relevant characteristics
Derivation
Non-replicating"
pOJ260
3.5
pSETI51
6.2
pKC1132
3.5
Am R rep pUc
Th RAp R .,cylE + rep ptjc
Am R rep pUc
pKCII38
pKCI250
Am a xylE + rep ptrc
Sp a rep pl'sa
See Fig. 1
See Fig. 1
pOJ260; deletion of
Kpnl to S p e l sites
See Fig. 1
See Fig. 1
Replicating ~'
pKC1139
6.5
pKCI218
5.8
pOJ446
10.4
Am R lacZ~ MCS rep t~
Am R lacZ~ MCS rep sop'°
Am R (cos)3 ~ rep scp'-°
See Fig. 2
See Fig. 2
See Fig. 2
Integrating ~'
pSETi52
5,5
pOJ436
11.1
pOJ444
35.3
pKCI116
10.1
pKC1163
5.5
Am t~ ktcZ~ MCS rep r'l~c'
Am n~(cos)3; rep pl ft.
Am w Km w rep v~tv' rep ~'tl
Am I~ (cos)3~rep ~'t't"
Am I~ hu'Z~ MCS rep t~''r
See Fig. 3
See Fig. 3
See Fig. 3
pOJ436 A a t l l deletion
_"~-~c-~-Fig. 3 legend
6.6
3.8
" Additional features present in these plasmids are a MCS in htt'Z~ and
oriT.
h Additional features present in these plasmids are oriT and rt,p r'vc.
Additional features present in th~;.se plasmids are oriT and im '~t'~v.
p
RESULTS AND DISCUSSION
(a) Plasmid constru,'tions
Table I lists plasmids described in this report. They represent three differ,,nt types: non-replicating plasmids
(Fig. 1), replicating plasmids (Fig. 2) and integrating plasmids (Fig. 3). They all contain the 760-bp oriT fragment
from RK2 (Guiney and Yakobson, 1983) and E. coli replication functions from pUC (Yanisch-Perron et al., 1985),
PI5A (Chang and Cohen, 1978), or PI (Sternberg, 1990).
Thus, these plasmids serve as cloning vectors for constructions to be made in E. coli, which subsequently can be
transferred by conjugation to the desired Streptomyces spp.
recipients.
Fig. 1 shows four plasmids (pOJ260, pSETI51,
pKC1138 and pKCI250) that do not replicate in Streptomyces spp., and thus they should be useful for gene disruption and gene transplacement experiments (Kuhstoss
et al., 1989; Muth et al., 1989; Richardson et al., 1990). For
cloning purposes in E. coil, these plasmids contain MCS
within lacZ~ which allows for easy detection of recombinant clones (Sambrook et al., 1989). Selectable markers
conferring Am R or Sp R were incorporated into pOJ260
(AmR), pKC1138 (Am R) and pKC 1250 (Sp R) because they
function in E. coil and Streptomyces spp. (Kuhstoss et al.,
1991; Howell and Rao, 1992). The Ap e marker for selection in E. coil and the Th R marker for selection in Strepto-
myces spp. were incorporated into pSETI51. Plasmid
pKC 1250 was constructed to facilitate gene disruption experiments in strains already containing integrated pUCbased plasmids. The use of, the Pl5A-derived replicon as
in pKCI250 will prevent recombination between homologous vector sequences.
To simplify the screening for segregants that have undergone gene transplacement, we inserted the xj,IE reporter
gene into pKCI138 and pSETI51, which is driven by a
synthetic promoter in pKC 1138 and by the er,nE promoter
in pSETI51. Although the xylE reporter gene from
Pseudomonas has been used in several Streptomyces strains
(Zukowski et al., 1983; Ingrain et al., 1989; Guthrie and
Chater, 1990; Bruton et al., 1991; Kuhstoss and Rao, 1991),
its expression in S. fradiae, when encoded by a single gene
copy, can be somewhat variable. Nevertheless, we have
used this reporter gene successfully for phenotypic discrimination between xylE ÷ and xyiE- to screen for plasmid
loss.
The plasmids shown in Fig. 2 contain replication functions for Streptomyces spp. and thus they can exist as autonomous, multicopy plasmids. These plasmids can be used
for complemt~nting mutations, increasing the copy number
of cloned genes and allele replacements. Plasmid pKC 1218
was derived from pOJ260 (Fig. 1) by inserting the SCP2*
replicon (Larson and Hershberger, 1984; 1986). Plasmid
pKC1139 contains a temperature-sensitive replicon from
45
(BamHllBg,I,)
Dral (BoIIIIBamHI)
-" /
_ (BamHllBcll)
Aatll ~ A ~ - a t l ,
$ma,
NdeNy/~
l
~~/Nde~
(BamHII~~yiE
katlf~lfd'
Kpnl.~=~ ~
x.o,
Eoo.,,
IL~ /IS~a-ci "" I
lacZ~'
I~'."
I
oSE1r151
• ~ I~"~',-I
..,,,-
I
Nde~a~
Ore~~l
l
Ira/
--
SacI
(ScallDral)
I I^,,,
| ....
\ ~,_
\3"
pKC1138
(e.ekbl
• I~ I Xbal"
I~ i BamHI"
reppUCJ ~ IS_at!!
" / ~lBssHll
_ /
~l EeORV°
~
Sacl
Clal"Sacl Xhol
~'~
repplSA/ Sspl
Nhel
Fig. 1. Restriction maps of pOJ260, pSETI51, pKC1138 and pKCI250. Plasmid pOJ260 is a derivative of pKC787 (Kuhstoss et al., 19911. The 0.8-kb
re'iT fragment was excised frona pPM803 (Mazodier et al., 1989) on a Pstl fragment and was subcioned into the Nsil site of an intermediate plasmid,
pKCI055. This plasmid contains cloning sit¢.s for Bglll, Nsil, Spei and BamHl, and thus the cloned oriT fragment could be excised with Bglll BamH!
and inserted at the Bglll site in pKC787. (BamH!Bglll) indicates the junction created by iigating BamH! generated ends to Bglll-generated ends. Plasmid
pSET151 was derived from pHJL401 (Larson and Hershberger, 1986) by deleting the SCP2* replicon on a Bell fragment and inserting a cassette containing oriT and the xrlE structural gene driven by the ermE promoter (p ......~..)(Bibb et al., 1985). The oriT was obtained from pKCI055 on a BamHlBglll fragment (as described above). The Bglll-generated end was ligated to the BamHl-generated 3' end of a BamHl to Bglll fragment containing the
xy!E structural gene (Kuhstoss and Rao, 1991). Promoter p,.,,,~: was isolated from pKC488 (Stanzak et al., 1986) on a BamHI-Kpni fragment, and
subc!oned into corresponding sites in p u c I g . The cloned fragment was excised on a BamHI-EcoR! fragment such that the BamHl-generated 3' end
could i~e ligated to the Bg/ll-generated 5' end of the xylE-containing fragment, yielding a p,.,.,,~::xylE::oril" cassette which was ligated to the Bcll fragment of pHJL401. The resulting linear fragment ~vas circularized after the 5' extensions of the EcoR! site of the cassette and the Bell site of the vector
were filled ih~ wkh Pollk. Piasmid pKC1138 is a derivative of pKC787, in which the region between the Bglll and Clal sites was replaced with a cassette containirg u'IE and oriT: This cassette consists of a 1.2-kb Dral-BamHl fragment encoding the xylE structural gene (Kuhstoss and Rao, 1991)
and a synthetic promoter sequence (5'-CTAGTAGGCTTGACACTTTATGCTTCCGGCTCGTATAATGTGTGCATA-3'), which was ligated to a
2.7-kb Cla l-Sea I fragment containing oriT from pS U P202 (Simon et al., 1983)via the Dra l-generated end and the Sea l-generated end, respectively. Plasmid
pKCI250 is a deri,'ative of pKC978 (tlowell and Rao, 1992). The 0.8-kb NheI.Sspl fragment contains the PISA ori (Chang and Cohen, 1978), and the
1.5-kb Xmnl-Nhel fragment, ligatcd between S,~pl and Nhel sites, cont'.,ms Sp I~ and was obtained from pHP4512 (Prentki and Krisch, 19841. Asterisks
indicate known unique restriction sites in the lacZ,~ MCS.
S. ghanaensis (Muth et al., 1989) which functions only at
temperatures below 34 ~C. Thus, this plasmid constitutes a
useful delivery system for transposons (Solenberg and
Baltz, 19911 and for experiments requiring homologous recombination between plasmid-borne sequence and the
chromosome. The cosmid vector pOJ446 contains the
SCP2* replicon and is capable of accepting up to approx.
35-kb DNA segments. The BamHl cloning site in pOJ446
is flanked by two restriction sites, Xbal and Spel, that are
rare in Streptono,ces spp., thus allowing for easy removal of
the intact cloned DNA. The T3 and T7 bacteriophage promoters were added to facilitate transcriptional analyses
(Loewy et al., 1989).
Plasmids and cosmids that can integrate site-specifically
at the ¢C31 attachment site are shown in Fig. 3. They give
rise to stable exconjugants that can be propagated without
detectable loss of plasmid markers, even in the absence of
drug selection (E. T. S., unpublished). The plasm!d vectors
contain the Am R marker for selection and lacZ~ with MCS
for cloning DNA. The cosmid vectors contain three cos
sites (Rao et al., 1987; Richardson et al., 1987) for in vitro
DNA packaging. Unlike the non-replicating vectors, these
vectors can be used to insert homologous or heterologous
DNA into the chromosome at the unique attB site (Kuhstoss and Rao, 1991). The ¢C31-driven, site-specific recombination is apparently very efficient because plasmids
containing as much as 8 kb of homologous DNA were
found only at the ¢C31 att site, with no detectable integration by homologous recombination (E. T. S., unpublished).
An E. coli c!~,tng vector (pOJ444) utilizing the bacteriophage P1 packaging system has also been constructed,
which potentially could be used for cloning DNA fragments of approx. 100 kb (Sternberg, 1990; 1992). Although
this plasmid has been shown to integrate site-specificaUy at
46
HIIBglII)
F
/
/
Pall---/
] .....
[ '"P'~
Kpnl-'~
\
~
/Bglll
orlT ~y.~
A Hlndlll
~t \ / I Pstl
lacZ~ ~( [Sail
pKC1'139
• I~ I XbaP
-(6.Skb)
I~ [ BamHI'
rePPuc ] ~ I SaclI
"
_
\
/ W
Clal Sacl Xhol
/
/
Xbal"
\ I,,,rr
\ '~g~'-
~1 B s s H l I
.......
\ ~.
~1ECORV"
_. . . . . / \ I SacZ!
reP'"~/ \IBssHII
/
\1ECORV*
~ EcoRI °
Sacl
Sacl Xhol
Sacl
Kpnl Sphl Pvull
XhoI~.~ 7 ,
""
V/NheI
7 L.
Sael-.../ / .... " ,,~aaaA
P"tx'-t t_.,. ~.11iq
\~, ....
- .......
~Z-Clat
Ik"--lXbal
I \ IEcoRVI
___
/
\lBamH]'l
Psll
Oral-~
Oral
cos ~os
~
/
I
Dral Dral
//~Pvuh
BOtH
Hpal
Fig. 2. Restriction maps of pKC1139, pKCI218 and pOJ446. Plasmid pKC1139 was derived from pOJ260 (Fig. 1). The 2.9-kb region between the Clal
site and oriT containing the temperature sensitive replicon from pSW344E was excised with Pndl+Xhol (Muth et al., 1989), and inserted between the
Kpnl and Spel sites in pOJ260 after treatment with T4 DNA polymerase. Plasmid pKCI218 is a derivative of pOJ260 (Fig. 1). The 2.2-kb BamH! fragment
from pHJL366 (Larson and Hershberger, 1986) containing the SCP2* replicon was inserted at the Bglll site (BamHl/Bglll indicates the two junctions)
in pOJ260. Plasmid pOJ446 was derived from pKC505 (Rao et al., 1987) by a series of steps. The unique BamHl site can be used to insert DNA fragments, and subsequent cutting a Pvull or Hpal generates the two arms used in cosmid cloning (Rao et al., 1987). To generate pOJ446, oriT was isolated
from pPM803 (Mazodier et ai., 1989) on a Pstl fragment which was inserted at a Pstl site. Asterisks indicate known unique restriction sites in the lacZ~
MCS in plasmids pKC1139 and pKCI218.
the ¢C31 art site in S. fradiae, S. ambofaciens and Streptomyces fividans, we have not yet tested it with large DNA
inserts.
(b) Conjugal transfer of vectors from Escherichia coli and
Streptomyces spp.
Conjugal transfer of DNA from E, coli to Streptomyces
spp. requires the 760-bp oriT fragment and transfer functions supplied in trans by the E. coil donor strain S17-1
(Mazodier et al., 1989; Simon et al., 1983). Most of our
experiments were done using S.fradiae as the conjugal
recipient, because PEG-mediated protoplast transformation of S. fradiae occurs with very low efficiency, particularly when large plasmids or cosmids isolated from E. coli
are used. Although highly transformable strains of S.fradiae have been isolated following several rounds of mutagenesis, these strains were constructed in tylosin nonproducers (Matsushima et al., 1987) and appear to have
acquired additional mutations that limit their use. Another
strain, Sa. spinosa (Mertz and Yao, 1990), was refractory
to PEG-mediated transformation and the only successful
means of introducing DNA was by conjugal transfer from
E. coil
Conditions used for conjugal matings between E. coli
and S. fradiae are given in the legend to Fig. 4. The origi-
nal protocol for E. cofi-Streptomyces spp. conjugal matings
reported by Mazodier et al. (1989) used freshly germinated
spores of the streptomycete as the conjugal recipient. We
have used this protocol successfully, but in order to accommodate actinomycetes that sporulate poorly (or not at
all) and to expedite the preparation of the recipient culture,
we have developed a protocol using vegetative mycelium
rather than spores. Using pSET152 (Fig. 3), we have reproducibly achieved exconjugant frequencies of 10-15 % of
the initial viable recipient population using either germinated spores or vegetative mycelia. Similar results were
obtained with pSETI69 (not shown) which contains the
xylE gene driven by the O,IF promoter (E. T. S., unpublished). Although exconjugants have been obtained from
matings carried out at temperatures between 29°C and
37°C, 37°C appears to be optimal, yielding about three
times as many exconjugants as obtained at 29°C. Matings
done on AS 1 medium (Baltz, 1980) generated exconjugants
at a frequency 1000-fold greater than on TS agar; no exconjugants were obtained on modified R2 medium (Baltz,
1978).
Plasmid size affected conjugation frequencies. In general, the frequency was much higher (100-1000 times) with
small plasmids (approx. 5.5 kb) than with large (approx.
20 kb) plasmids or cosmids (45-50 kb). However, the pre-
47
HindIII Aatll* EcoRI
AatlI*/'' / /
Hindlll
Clal,~" / ~ . . ~
Ncol
EcoRI~
i~o~--"~ /HindIlI
SacI~
""
",(
Sail HindII!
~
/
/intoC31
/ ....
AatII /
S hi \ I
P ~
~
/IPstI
\
/ I sa"
I I \ / IXbaI*
,. . . . . ~ ~/
..... i ~
oSET152
" (5.5
- kb)
Xhol---.//
Sacl-.~//AmR
XmnI~;:~ .~_ ._
IBamHI"
I SaclI
l\ IBssHII
/ ~ I EcoR~"
,s,i\
-,,
PStl~
\ /ISpeI I
pOJL4~, 1"7,! ~ IDra~mHl~
,,,.,-,
,3,
lvOra:
^°"
I
I
Oral---~';~'~cos--¢'/ Sail
_, . ~ -n- yu~
/C,--DraI
Dral n " ~" . . ~ \ P v u ~
~-Dra] D~al Hpa]BglI!
q'"Y~,~
s'=i/
\
sac,
Bali ApaI Xhol
EcoRI
/ /
_,r r
'~"" - ~....sal]
,ox, " " ' "
,p
oo,,
'"t°~'7~ 'cfa~
Ec°R'r \ ~
P1oac IOXP~'EcoRl
~,~,,
clal
o,L,"am""
Seal
....
Fig. 3. Restriction maps of pSETI52, pOJ436 and pOJ444. Plasmid pSETI52 was derived from pKC912A16 (Kuhstoss and Rao, 1991) by deleting the
1.5-kb Aatll fragment and by changing the sequence of the EcoRl site adjacent to the ¢C31 att site to 5'-AAATTC by site-specific mutagenesis. The
0.8-kb oriT-containing Pstl fragment from pPM803 (Mazodier et al., 1989) was initially subcloned into pUCI9. The cloned fragment was excised with
Xbal and Hindlll and treated with Pollk fragment of D N A polymerase to fill in the 5' extensions. The resulting fragment was cloned into similarly treated
Nhel site, which was regenerated at the Xbal/Nhel junction. Asterisks indicate known unique restriction sites in lacZ~ MCS. A related plasmid pKC1163
(not shown) differs from pSETI52 in that the regions between the Sphl and Pstl sites on one side of ariT and the Pstl-Ball sites on the other side of
or/T were deleted using Exolll nuclease, in pKC! 163, the EcoRl site in int *c'~' is present. Piasmid pOJ436 was derived from pKC731 (Kuhstoss et al.,
1991). The region in pKC731 between Kp,] and Bam HI was replaced with a 93-bp synthetic DNA fragment 5'-GATCTCTAGATTAATTGGGAGTGATI'TCCCTTG'ITTAAAGGATCCTI'TAAATCGCCCTATAGTGAGTCGTATTACAATTCACACTAGTGGTAC-3'
which created a unique
BmnHl cloning site flanked by Dral sites and T7 and T3 promoter sequences. Flanking the promoters are additional restriction sites for Spel and Xbal.
Deletion of the small Aatll fragment from plasmid pOJ436 generated plasmid pKC! i 16. Piasmid pOJ444 is a derivative ofpNS582tetl4ADl0 (Sternberg,
1992) into which the oriT fragment from pPM801 was inserted at the unique Sail site. In addition, a 6-kb BmnHl-Bgill fragment encoding Am R and
i,t "pc3~ was inserted at the unique BmnHl site regenerating a unique BamHl site for cloning. This fragment was obtained from pKC438 (unpublished),
which is a derivative of pKC787 (Kuhstoss et al., 1991) containing the BamHi-Pstl fragment encoding Am R and int ~c31 from pKC731 (Kuhstoss et al.,
1991) in the MCS allowing for its excision with BmnHl+Bglll. Cutting the plasmid pOJ444 at BamHl and Seal sites generates the two fragments for
P! cloning.
cise numbers varied with the type of plasmid used and the
size and source of the DNA insert. This is not surprising
because recipients are expected to restrict incoming DNA
to varying degrees, depending on the frequency and sequence context of susceptible restriction sites.
S. ambofaciens can be readily transformed with plasmids
isolated from E. coli (Matsushima and Baltz, 1985), but
introduction of DNA by conjugal transfer provided an alternative and easier method. Most of the conditions and
variables described for S. fradiae apply to S. ambofaciens.
Piasmids with the oriT region could be mobilized, not only
from E. coli S17-1 where RP4 is chromosomaily located,
but also from E. coil strains in which RP4 is free in the
cytoplasm. With Sa. spinosa, we found that the mating
conditions described yielded exconjugants, although the
frequencies were considerably lower (1-200 per mating
plate) compared with S. fradiae or S. ambofaciens.
Using these conditions and the vectors described in the
report, we have created gene disruptions and gene replacements within the tylosin and spiramycin gene clusters in
S. fradiae and in S. ambofaciens (in preparation), increased
the gene dosage of the tylF gene in S.fradiae (E.T.S., in
prepara:ion), and introduced large (approx. 35 kb) homologous or heterologous DNA fragments on cosmid vectors
into S. fradiae, S. ambofaciens and Sa. spinosa (M.B., unpublished).
(c) Conclusions
(I) Non-replicating, freely replicating and integrating
plasmid or cosmid vectors were constructed and shown to
transfer efficiently from E. coil to Streptomyces by conjugation.
(2) The highest numbers of S. fradiae exconjugants were
obtained in matings carried out at 37 °C on AS 1 agar plates
using either freshly germinated spores or early exponential
phase mycelial fragments as the conjugal recipient.
48
Fig. 4. Conjugal transfer of plasmids from E. coil to Streptono'ces. One ml of a frozen mycdial culture of S. fradiae was diluted into 9 ml of TS broth
(Baitz, 1978) and grown for 18 h aerobically at 29°C. The culture was homogenized (Baltz, 1978) and 2 ml was transferred into 18 ml of fresh TS broth
and grown for 16 h at 29°C to obtain a late log phase culture. This culture was homogenized and fragmented with ultrasound (Baltz, 1978), and 1 ml
was transferred to 9 ml TS broth. The culture was incubated aerobically at 37°C for 3 h. The mycdium was recovered by centrifugation, washed once
in TS broth and resuspended in 2 ml TS broth (recipient culture). The E. coil donor, S17-1[pSET169], was grown at 37°C overnight in TY broth plus
100/ag Am/ml, subcultured 1:100 and grown for 3 h at 37 ~'C. The cells were pelleted, washed once in TS broth and resuspended in 2 ml TS broth (donor
culture). Equal vols of the donor culture and ten-fold serial dilutions of the recipient culture were mixed, and 100 ~d were plated to AS l (Baltz, 1980),
supplemented w!th 10 mM MgCI_,. Plates were incubated at 37°C lbr 16 h, and then covered with 3-4 ml of soft R2 agar (Baltz, 1980) containing !.5 mg
nalidixic acid and 1.5 mg of Am. Incubation at 37 °C was continued for about a week to allow outgrowth of the exconjugants, a, 50 #1 of the recipient
culture alone selected for Am resistance (negative control); b, 10 ld of recipient culture, no Am; e, 10 ld of a 10"~dilution of the recipient culture, no Am;
d, Am R exconjugants obtained when undiluted recipient was used; e, Am R exconjugants obtained when 10"~diluted recipient was used. No colonies grew
when 100 ld of donor culture was plated with Am and nalidixic acid. Incubation at 34°C instead of 37~C gave similar results.
ACKNOWLEDG EM ENTS
We are particularly thankful to P. Mazodier, R. Petter,
C. Thompson and J. Davies for sharing pPM801 and
pPM803 plasmids and E. coli S17-1 strain with us, without which this work could not have been done. We thank
M. Howell, M. Richardson, S. Kuhstoss, M. Basinski and
F. Laquier for sharing their experiences with some of the
plasmid vectors described in this paper. We also thank
Lilly Research Laboratories for supporting this work, and
Barbara Fogleman for typing the manuscript.
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