Journal of General Microbiology (1 986), 132, 207 1-2078.
Printed in Great Britain
207 1
Properties of in uitro Recombinant Derivatives of pJV1, a Multi-copy
Plasmid from Streptomyces phaeochromogenes
By C . R. B A I L E Y , ’ * ? C . J . B R U T O N , * M . J . B U T L E R , ’ $ K . F. C H A T E R , 2
J . E . H A R R I S ? $ A N D D . A. H O P W O O D 2
I Beecham Pharmaceuticals UK Division, Clarendon Road, Worthing,
West Sussex BN14 8QH, UK
?John Innes Institute, Colney Lane, Norwich NR4 7UH, UK
(Receiced 14 January 1986)
The 10.8 kb plasmid pJV1, isolated from Streptomyces phaeochromogenes, has a high copy
number (about 150) and a broad host range among Streptomyces spp. Several pJVl derivatives
carrying the thiostrepton resistance gene (tsr) of S . azureus were made. One derivative,
pWOR191, was shown to promote its own transfer and to mobilize chromosomal markers in S.
lividans. Another derivative, pWORlO9, was non-transmissible. Deletion in vitro of a segment
of pWOR109 gave pWOR120 (5-6kb), which has single BamHI and BgnI sites shown to be
capable of accepting ‘foreign’ DNA such as a previously cloned S . antibioticus DNA fragment
encoding tyrosinase, giving vectors (pWOR125, pWOR126) with properties resembling the
well-established multicopy vector pIJ702. Shuttle vectors capable of functioning in both S .
fividuns and Escherichia coli were also constructed. The region of pJVl essential for replication
and maintenance was localized to a 2-5 kb segment. Stable maintenance of pWOR109 and
pWOR120 was observed in the presence of derivatives of pIJ101, the progenitor of pIJ702.
INTRODUCTION
Doull et al. (1983) described a multicopy plasmid, pJV1, present in Streptomyces
phaeochromogenes NRRL B3559. Independently, we also discovered this plasmid, and were
interested to study it because at the time we knew of only one other high copy number
Streptomyces plasmid, pIJlOl (Kieser et al., 1982), though several others have since been
described (reviewed by Hopwood et al., 1986). Moreover, the S . phaeochromogenes cluster, as
studied by numerical taxonomy (Williams et al., 1983), is not closely related to the S . griseoruber
cluster which contains S . lividans ISP 5434, the organism from which pIJlOl was originally
isolated. A comparison of pIJlOl and pJVl might, therefore, reveal generalities about
Streptomyces plasmids (notwithstanding the observed wide host range of many Streptomyces
plasmids: Hopwood et al., 1986). It was also possible that pJV1, like pIJ101, might form the
basis of a useful set of cloning vectors to add to the ‘families’ already in use (reviewed by Chater
et al., 1982; Hopwood & Chater, 1982; Bibb et al., 1983). This paper describes the physical
characterization of pJV 1, the construction of derivatives carrying the thiostrepton resistance
gene (tsr) first cloned from S . azureus by Thompson et 01. (1980, 1982b), and the use of these
derivatives both in the initial characterization of some genetic aspects of the plasmid and as
cloning vectors.
t Present address:
Apcel Limited, 545 Ipswich Road, Slough, Berks SLI 4EQ, UK.
$ Present address: Pfizer limited, Ramsgate road, Sandwich, Kent CT13 9NJ, UK.
tj Present address: Food Research Institute, Colney Lane, Norwich NR4 7UA, UK.
0001-3206 0 1986 SGM
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C . R. B A I L E Y A N D O T H E R S
METHODS
Bacterial strains and plasmids. pJV 1 was isolated from S . phaeochromogenes NRRL B3559. S . lividans 66 (John
Innes Institute Stock no. 1326: Lomovskaya et al., 1972) was used as the standard recipient organism for
recombinant plasmids. Genetically marked S. lividans strains lacking endogenous sex factors, used for
recombination studies, are described in Table 1. Other Streptomyces species are given in the text; those used for
host range studies are listed in Table 2. pIJ702 was described by Katz e f al. (1983). E. coli DHI (Hanahan, 1983)
was used as host for pAT153 (Twigg & Sherratt, 1980) and bifunctional plasmids.
Culture conditions and transformationprocedures. YEME, a yeast extract/malt extract medium, and R2YE, an
agar protoplast regeneration medium containing yeast extract, were as in Thompson er al. (1980). Protoplast
formation and transformation of S. lividam were as in Thompson et al. (1982a). Direct selection for thiostreptonresistant transformants was made by overlaying the regeneration plates (R2YE) with 2.5 ml soft Nutrient Agar
(Difco) containing 500 pg thiostrepton ml-' after incubation for 20 h (Kieser et al., 1982). Thiostrepton (kindly
donated by Mr S. J. Lucania of E. R. Squibb and Son, New Brunswick, NJ, USA) was used in liquid and solid
media at 50 pg ml-'. E. coli DHI was transformed by standard procedures (Maniatis et al., 1982); ampicillinresistant transformants were selected on Luria agar (Miller, 1972) containing 50 pg ampicillin ml-'.
DNA isolation and in vitro manipulation. Plasmids were isolated by methods described by Kieser (1984) and
Hopwood e f al. (1985). Rapid preparations of E. coli plasmids were carried out by the boiling method (Holmes &
Quigley, 1981). Restriction enzyme digestion, bacterial alkaline phosphatase treatment, agarose gel electrophoresis and ligation of DNA were essentially as in Maniatis et al. (1982).
RESULTS
Initial characterization of pJV1
In the course of an experiment unconnected with plasmid isolation, total DNA of S .
phaeochromogenes was prepared by a method that did not specifically aim to preserve the
conformation of supercoiled covalently closed circular (CCC) molecules. Agarose gel
electrophoresis of this DNA revealed a conspicuous sharp band migrating ahead of the usual
diffuse band of linear, predominantly chromosomal, DNA fragments. Electrophoresis in a
second dimension after UV irradiation (Hintermann et al., 1981) showed that the band
contained supercoiled CCC DNA. The plasmid was then isolated from S.phaeochromogenes by
CsC 1/ethidium bromide centrifugation for further study. A restriction map of the plasmid, at
that time designated pIJ109, was determined by analysis of single and double digests (Fig. 1).
Comparison with a less detailed map (Doull et al., 1983) of a plasmid independently isolated
from the same S.phaeochromogenes strain leaves no doubt that the two plasmids are the same,
and we have, therefore, adopted the designation pJVl used by Doull et al. (1983). The plasmid
consists of 10.8 kb, and 48 h YEME or Difco Nutrient Broth cultures had an apparent copy
number of about 150 per chromosome estimated by micro-densitometric analysis of
photographic negatives of ethidium bromide-stained agarose gels after electrophoresis of
different dilutions of total S. phaeochromogenes DNA preparations (Kieser et al., 1982).
Construction of pJVl derivatives carrying the tsr gene
DNA of pJVl was partially digested with MboI such that much of the DNA was cleaved once
to yield the full-length linearized molecule while a proportion of the DNA was cleaved further to
yield partially deleted molecules. As a source of the tsr gene, pIJ702 (Katz et al., 1983) was
cleaved at its four Bcfl sites and treated with bacterial alkaline phosphatase to suppress
religation of the fragments. The two digested DNA preparations were mixed, digested with
XhoI (which cleaves pIJ702 within its replication origin region, but does not cleave pJVl), and
used to transform protoplasts of S. lividans. Of 200 thiostrepton-resistant transformants
obtained, 199 were white (Mel-) and presumably contained recombinant plasmids of the kind
sought. The white transformants were systematically screened for plasmid DNA and some of
the recombinant molecules obtained were subjected to restriction analysis.
In two of the three derivatives shown in Fig. 1 - pWORlOl and pWOR103 - a segment of
pJVl was replaced by the tsr fragment without further rearrangement. In the other derivative,
pWOR191, the tsr fragment was inserted into an otherwise intact pJV1. Another derivative,
pWOR109 (Fig. 2), was evidently the result of a three-way ligation between the tsr fragment and
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Derivatives of streptomyces plasmid pJV1
2073
Distance from iEcoRI site (kb)
1. 0
13. 4.9
14. 5.2
2. 0-15
15. 6-55
3. 0.4
4. 1.55
16. 6-95
5 . 1.9
17. 7.15
6. 2.3
18. 7.25
7. 2.4
19. 8.7
8. 2-45
20. 9.4
9. 3.85
21. 9.45
10. 4.3
22. 9.5
11. 4.6
23. 10.5
24. 10.65
12. 4.7
1. 10.8
Fig. 1. Restriction map of pJVl. No sites were found for HindIII, PsfI, XhoI, XbaI, ClaI, Ben, HpaI and
SphI. (SphI sites were absent from pJVl sequences tested in recombinant plasmids propagated in E. coli
or S . licidans, eliminating the possibility that modified SphI sites were present in pJVl propagated in
the SphI-producing S.phaeochromogenes.)The position of insertion and the orientation of tsr are shown
for pWORlO1, pWOR103 and pWOR191. The deletions of pJVl D N A associated with construction of
pWORlOl and pWOR103 are indicated by the bars.
Distance from EcoRI site (kb)
1.
24.
23.
22.
6.
7.
8.
9.
0
0.15
SSfI
0.3
Smd
1.3
BamHI 2-6
SmaI
2.7
BglII
2.75
SStI
4.15
EcoRI
SSfII
10.
11.
12.
13.
14.
15.
PUUII
SstII
SSfI
KpnI
SmaI
SsfII
3. KpnI
2. S S f I
1. EcoRI
4.6
4.9
5.0
5.2
5.5
6-85
7.6
1-85
8.0
Fig. 2. Restriction map of pWOR109. The arrowheads show the orientation of the two composite pJVl
fragments and fsr.Restriction site numbers are as in pJVl (Fig. 1). The bar indicates the KpnI fragment
deleted to give rise to pWOR120.
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C. R . BAILEY A N D OTHERS
Table 1. Chromosomal recombination in crosses between S . lividans strains, mediated by
p WOR191
Strain numbers
of parents*
TK54
TK54
TK54(pWOR191)
TK64(pWOR191)
3224
3224(pWOR191)
3224(pWOR 19 1)
x
x
x
x
x
x
x
TK64
TK64(pWOR191)
TK64
TK54(pWOR191)
3225(pWOR191)
3225
3225(pWOR 191)
Average recombinants
per sum of parentalst
<4
3.6
7.1
<1
6.0
4.6
1.3
x 10-91
x
x
x 10-8
x
x
x 10-75
* TK54 his-2 leu-2 spc-1; TK64 pro-2 str-6; 3224 his-2pro-2 spc-1; 3225 leu-2 str-6. All four strains are
SLP2-SLP3- (Hopwood et al., 1983).
t Average on media selecting his+/spc or pro+/spc.
1Data from Kieser et al. (1982).
6 Of which 1 in 10 are definitely recombinants (differ from both parents by two markers).
two non-overlapping MboI fragments of about 2 kb and 5 kb, in an inverted orientation
compared to their configuration in pJV1. These results show that the region essential for
maintenance lies in the 5 kb between sites 6 and 15.
Plasmid transfer and ability to promote chromosomal recombination
The lethal zygosis reaction (Ltz) caused by Streptomyces plasmids is correlated with plasmid
transfer (Hopwood et al., 1983). We could not detect pocks in regenerating S . lividans lawns after
attempted transformation with pJVl DNA. However, the set of pJVl derivatives generated in
the experiment described in the above section were tested for their Ltz phenotype, by replication
of sporulating patches of S . lividans containing the pJV 1 derivatives onto a lawn of 1326. Weak
Ltz zones at the junction of the cultures were seen in some cases, but pWOR191 (Fig. 1)
appeared to elicit a particularly strong reaction.
Many derivatives, including pWORlO1, pWOR103 and pWOR109, did not give a visible
reaction, indicating a possible defect in transfer functions. In the case of pWOR109, transfer
deficiency was directly demonstrated by analysis of mating mixtures [TK54(pWOR109) x
TK641; between 10-6 and 1O-* of the recipients acquired thiostrepton resistance in five crosses,
compared with transfer frequencies of 48-88% for pWOR191.
The fertility properties of pWOR191 in S . lividans were studied in the test system described by
Hopwood et al. (1983). Strains carrying pWOR191 were prepared by transformation, and the
crosses shown in Table 1 were carried out. The results show that pWOR191, when present in one
parent, promoted chromosomal recombination. The average frequency of 5 x 10-6
recombinants per sum of parentals was at the lower end of the range of frequencies ( 5 x
to 5
x
reported for other plasmids (Hopwood et al., 1983). Many times fewer recombinants
were detected in plasmid+ x plasmid+ than in plasmid+ x plasmid- crosses, indicating ‘entry
disadvantage’ for the plasmid (Bibb & Hopwood, 1981; Hopwood et al., 1983).
Host range of pJVI
The host range of two tsr-containing derivatives, pWOR109 and pWOR191, was determined
by protoplast transformation, using a Streptomyces series similar to that used by Kieser et al.
(1982) to examine the host range of pIJ101. Selection for thiostrepton resistance was made by
overlaying the regenerating protoplasts after 20 h with soft agar containing the antibiotic. The
results are shown in Table 2. Successful transformation was confirmed in each case by isolation
of plasmid DNA of the correct size from at least one thiostrepton-resistant colony, except in the
case of S . kanamyceticus NRRL B2535 where thiostrepton-resistant colonies were obtained on
transformation with pWOR109 and pWOR191 but no plasmid DNA could be isolated.
N o attempt was made to optimize protoplast formation, transformation and regeneration
conditions for each species; the procedures developed for S . lividans were used throughout
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Derivatives of’streptomyces plasmid pJVl
Table 2. Transformation of various streptomycetes by p WOR109* and p WOR191*
Transformation by
c
Strain
p
A
-
pWOR109
S. alhus G R-sal-2 ilv-1:
S. albus P CMI 52766
S . coelicolor A3(2)
S . fradiae NRRL B3357
S . glaucescens ETH 22794
S . griseus ATCC 10137
S . licidans 66
S . parculus ATCC 12434
S . pristinaespiralis ATCC 25486
S . rimosus NRRL 2234
S . fradiae ATCC 10745
S . cinaceus NCIB 8852
S . kanamycericus NRRL B2535
++
+
+
+
+
++
+
+-
\
pWOR191
+
+
+
+
+
+
+
+
+
+
+-
Transformation
frequency?
low
low
high
high
low
low
high
high
low
low
low
low
* pWOR109 and pWOR191 DNA prepared from S . lividans 66.
t Low, < lo3 transormants per pg plasmid D N A ; high, > lo3 transformants per pg plasmid
$ A restriction-deficient mutant described by Chater and Wilde (1980).
DNA.
(Thompson et al., 1980). The low transformation frequencies observed for many species could be
explained either by the use of non-optimal procedures or by host restriction systems. Differences
in apparent host range between pWOR109 and pWOR191 were seen only in cases of very
low transformation frequency and are of doubtful significance.
Compatibility of pJVl derivatives with other plasmids
Several of the strains used in the host range studies were known to contain autonomous and/or
integrated plasmids. Successful transformation of S. coelicolor, S . parvulus and S . glaucescens
showed that pWOR109 and pWOR191 were compatible with SLP1, pIJl10 and pIJ408, which
are present as stably integrated sequences in the respective strains (Hopwood et al., 1984).
(Compatibility with other resident S . coelicolor plasmids SCPl, SCP2 and SLP4, although likely,
cannot be assumed as the presence of these plasmids was not tested in the transformants.) The
phenotypically cryptic autonomous plasmid pSAl (Orekhov et al., 1981), however, was shown
physically to be present in transformants of S . albus G .
The question of the compatibility of pJV 1 and pIJ 101 derivatives was particularly important,
because of the pre-eminence of the latter as high copy number cloning vectors. S . lividans TK54
(Kieser et al., 1982)containing pWOR109 was transformed with pIJ58 (containing the essential
replication region of pIJ 101, and the aph gene of S .fradiae: Kieser et al., 1982)to give neomycinresistant colonies. One such colony was entirely non-selectively subcultured to give confluent
growth, which was harvested and re-plated again non-selectively to give single colonies.
Replication (despite being inefficient because the colonies did not sporulate very well) showed
that at least 87% of colonies were resistant to both thiostrepton and neomycin. Analysis of CCC
DNA from cultures derived from seven of the original transformed colonies, grown with and
without selection, showed that six retained both plasmids apparently unaltered and in high yield
in both selective and non-selective conditions. The seventh culture, grown non-selectively,
contained pWOR109 at a high level but only a trace of pIJ58. The latter result was consistent
with the known propensity of pIJ58 to be fairly easily lost during non-selective growth (Kieser et
al., 1982). Overall, the results showed that the two families of multicopy plasmids are
compati ble.
Use of pJVl derivatives in DNA cloning
The plasmid pWOR 120 was constructed by in vitro deletion of the small KpnI fragment (sites
13 to 3) from pWOR109 (Fig. 3 ) . In S . lividans, both pWOR109 and pWOR120 were inherited
stably, and their potential as cloning vectors was investigated. Sites for BamHI and BgflI are
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C . R . BAILEY AND OTHERS
Ssrl
pWOR123
pWOR I2 I
Fig. 3. Insertion of PAT1 53 and the me1 fragment into pWOR120. The orientation of pAT153 ( D ) and
indicated. The bar shows the essential region of the plasmid. Restriction site numbers are
from pJVl (Fig. I).
mu1 ( b ) are
useful for cloning with a variety of restriction enzymes including the widely used MboI, so
attention was principally focused on the single BamHI (6) and BglII (8) sites of pWOR120. The
E . coli vector, pAT153, was linearized with BamHI and ligated with BamHI-digested and BgnIdigested pWOR120, respectively. The hybrids were found in E. coli DHI by screening plasmid
DNA of ampicillin-resistant tetracyline-sensitive transformants. pWOR121 and pWORl22
have PAT153 inserted in the BamHI and BglII sites of pWOR120, respectively (Fig. 3). Both
these constructs could be introduced by transformation into S . liuidans by selection for
thiostrepton resistance. This experiment suggested that neither the BamHI nor the BgnI site of
pWOR120 was in an essential region, and that both were potential cloning sites.
In a further experiment, the BclI-generated tyrosinase fragment (me0 from pIJ702 (Katz et
al., 1983) was inserted, in both orientations, in the BamHI and in the BglII sites of pWOR120
(Fig. 3). These constructs, pWOR123, pWOR124, pWOR125 and pWOR126, gave the typical
black Mel+ phenotype in S . liuidans on R2YE medium supplemented with tyrosine. There was
no evidence of spontaneous loss of melanin production, so that the BglII and SphI sites of the mel
gene in these plasmids should be suitable for the insertion of foreign DNA, giving concomitant
insertional inactivation of melanin production, as in the widely-used vector pIJ702.
Since the EcoRI site of pJVl could be deleted without noticeable loss of plasmid stability (e.g.
in pWOR103) it was expected to be suitable for cloning EcoRI-generated fragments of foreign
DNA. Indeed, Ikeda et al. (1984) succeeded in introducing an EcoRI fragment (of DNA from
the phage 4C31KC550) that contained the S . uinaceus viomycin resistance gene (uph), the S.
coelicolor glucose kinase gene (glk), and parts of pBR322, into the EcoRI site of pWOR109.
However, the insert was subject to constant deletion, for reasons that are unclear. We tried to
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Deritiatives of' streptomyces plasmid p J V l
2077
introduce into this EcoRI site a fragment differing from that used by Ikeda et al. (1984) only in
lacking the glk insert. Although viomycin-resistant transformants were obtained, the plasmid
DNA isolated from them again contained nunierous deleted forms, showing that the problem
was not caused simply by the cloned glk gene. Finally, we tried to insert an EcoRI fragment of
the E. coli plasmid pSKSlOl (Shapira et al., 1983) containing the aphII gene of Tn.5 (known to
give rise to kanamycin resistance in S. lividans: Foster, 1982; Gil & Hopwood, 1983) into the
p WOR 109 EcoRI site. Kanamycin-resistant transformants were obtained, but again they
always contained complex plasmid populations, usually revealing only trace amounts of the
expected 2 kb aphll-containing EcoRI fragment on agarose gels of EcoRI digests.
Further localization of'the essential region of pJV1
The only region common to all pJVl derivatives described in this work was the 2-7 kb
segment including BamHI (6) to KpnI (13) of pJVl (Fig. 1). With the construction of the
bifunctional replicons pWOR121 and pWOR122, there was the opportunity to delete parts of
the Streptomyces DNA in vitro, check the constructions in E. coli, and subsequently attempt to
introduce the plasmids into Streptomyces. Such an experiment was carried out using pWOR121
(Fig. 3), which was cut with SstI and religated to generate a set of molecules based on the 8 kb
SstI fragment including PAT1 53 and tsr, and containing various deletions and reassortments of
SstI fragments. Plasmids lacking the SstI fragment, sites 9 to 12, were unable to replicate in S .
liuidans, but no other SstI fragment was essential. This result further narrows down the essential
DNA to the 2.5 kb region including BamHI (6) to SstI (12), shown in Fig. 3.
The essential nature of this region was also indicated by another experiment. Insertion of a
partial TaqI digest of wild-type pJVl into the ClaI site of the pBR322 derivative pIJ36
(containing the S . vinaceus vph gene encoding viomycin resistance; Thompson et al., 1982b)gave
one derivative, pIJ550, containing an apparently full-length copy of pJVl interrupted at a site
very close to SstI site 9. This plasmid gave no viomycin-resistant transformants with S. liuidans
protoplasts.
DISCUSSION
Like several other Streptomyces plasmids (Hopwood et al., 1986), pVJl contains a very short
segment (at most 2.5 kb) of essential DNA. Our experiments have not been sufficiently detailed
to allow rigorous allocation of functions to non-essential regions of the plasmid, though it seems
likely that at least some of the plasmid transfer functions lie in the common segment (sites 19 to
21) deleted in the transfer-deficient derivatives pWOR101, pWOR103 and pWOR109. It is also
possible that a gene (or genes) influencing transfer is located between sites 14 and 15 since
pWOR191, containing tsr inserted in this region, gave a greatly intensified pock phenotype,
conceivably by disturbing the normal balance of transfer-related functions.
The vectors described in this work have several useful properties. They have high copy
number, estimated at 150 per chromosome for pJV1. They are non-transferable, so that
superinfection of clones does not take place on protoplast regeneration. The presence of
available BamHI and BgnI sites will allow the cloning of Sau3AI (MboI) generated fragments.
The vectors pWOR125 and pWOR126, the single BgnI and SphI sites of which are both in the
me1 fragment, can be used in an analagous way to pIJ702 where inactivation of me1 expression is
a convenient means of scoring insertion (Katz et al., 1983). They exhibit a very wide host range,
and at least in the case of S.erythraeus, pWOR109 is apparently more easily used than is pIJ702
( C . R. Hutchinson, personal communication).
No examples of incompatibility of pJVl derivatives with other known plasmids have been
found. This co-existence of pJVl derivatives with other plasmids could be very useful in
examining the effect of combinations of independently cloned genes within the same cell,
without the need for further in uitro manipulation. The recent development of this and several
other alternative multicopy vector systems for Streptomyces (see Hopwood et al., 1986, for a
catalogue) can therefore be expected to enhance the value of the currently most-used plasmid
vector family, based on pIJlOl (Kieser et al., 1982) and including pIJ702 (Katz et al., 1983) and
several promoter-probe vectors (Mervyn J. Bibb, Maureen J. Bibb, T. Kieser and J. M. Ward,
personal communication).
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C. R . BAILEY A N D OTHERS
We wish to thank Nicola Hobden for her excellent technical assistance, Helen Kieser for carrying out fertility
tests, and Tobias Kieser for a gift of pIJ58 DNA.
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