Download Construction of a new cloning vector utilizing a cryptic plasmid and

FEMS Microbiology Letters 168 (1998) 195^199
Construction of a new cloning vector utilizing a cryptic plasmid
and the highly expressed melanin-synthesizing gene operon from
Streptomyces castaneoglobisporus
a
Institute of Pharmaceutical Sciences, Hiroshima University School of Medicine, Kasumi 1-2-3, Minami-ku, Hiroshima 734-8551, Japan
b
Department of Chemistry and Material Engineering, Ibaraki National College of Technology, Nakane 866, Hitachinaka,
Ibaraki 312-8508, Japan
c
Laboratory of Special Chemicals, Nippon Kayaku Co. Ltd., Iwahana-machi, Takasaki, Gunma 370-1208, Japan
d
Osaka University of Pharmaceutical Sciences, Takatsuki, Osaka 569-1041, Japan
Received 25 August 1998; received in revised form 16 September 1998; accepted 18 September 1998
Abstract
Streptomyces castaneoglobisporus HUT6202 overproduces a diffusible melanin pigment and harbors a cryptic 7.4-kb
plasmid, pHY6202. We constructed a Streptomyces cloning vector, pSY10CMM, consisting of the HUT6202 rep gene, the
thiostrepton resistance gene and an operon encoding the synthesis of melanin pigment, abbreviated mel, from S.
castaneoglobisporus. The vector, which has SphI and BamHI sites as cloning sites with insertional inactivation of the mel, is
a more convenient cloning vector than commonly used pIJ702, because of its broad host range for antibiotic-producing
Streptomyces strains and its much greater production of diffusible melanin pigment. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
Keywords : Cloning vector; Melanin pigment ; Streptomyces castaneoglobisporus
1. Introduction
Most plasmids found in the streptomycetes are
physiologically cryptic, except plasmids encoding
the production and resistance to methylenomycin A
* Corresponding author. Tel.: +81 (82) 257-5280;
Fax: +81 (82) 257-5284; E-mail: [email protected]
Abbreviations: Bm, bleomycin; mel, an operon encoding the
synthesis of melanin pigment; rep, the gene encoding a protein
essential for replication; Ts, thiostrepton
and pock-formation [1,2]. Several genes encoding the
antibiotic-resistant determinants have been cloned
from antibiotic-producing microorganisms using
pock-forming plasmids [2^4]. Genes cloned by these
plasmids have become useful markers in DNA technology to Streptomyces [5,6].
S. lividans ISP 5434 harbors four small high copy
plasmids, pIJ101 and its natural deletion variants
pIJ102, pIJ103 and pIJ104 [7]. A cloning vector
pIJ702, carrying the pIJ102 replicon, is commonly
used for Streptomyces gene manipulation [6]. This
0378-1097 / 98 / $19.00 ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
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Kayo Ikeda a , Koji Suzuki b , Hideki Yoshioka c , Katsushiro Miyamoto d ,
Tsutomu Masujima a , Masanori Sugiyama a *
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K. Ikeda et al. / FEMS Microbiology Letters 168 (1998) 195^199
2. Materials and methods
2.1. Plasmid and DNA fragment used for cloning
A cloning vector, pIJ702 [6] was used as a source
of Ts-resistance gene. A 3-kb BamHI fragment containing a bleomycin (Bm)-resistant gene, inserted
into pUC18 [11,12], was used to subclone into the
vector constructed in this study.
2.2. DNA manipulations
Plasmid from Streptomyces was isolated according
to the method of Kieser [13]. Preparation, transformation and regeneration of Streptomyces protoplasts
were carried out as described by Thompson et al. [5].
DNA sequencing was done by the dideoxy-DNA
chain-termination method [14] using an auto-sequencer (Applied Biosystems, USA).
3. Results and discussion
3.1. Construction of a cloning vector pSY10CMM
In this study, we found that S. castaneoglobisporus
HUT6202 harbors a high copy 7.4-kb plasmid, designated pHY6202. Fig. 1 shows the restriction map
of pHY6202. To develop the cryptic pHY6202 as a
cloning vector we subcloned the 1.05 kb BclI fragment containing Ts-resistance gene from pIJ702, into
the single BamHI site in pHY6202. After the S. lividans 1326 protoplasts, transformed with the chimeric
plasmid, were regenerated, Ts (50 Wg ml31 ) resistantcolonies were selected and analyzed for the presence
of the plasmid. The plasmid, designated pHY12, was
able to replicate in several antibiotic-producing
strains, including S. aureofaciens KCC S-0008, S.
griseochromogenes HUT6232, S. fradiae HUT6096,
S. kanamyceticus HUT6231, S. griseus 2247, Streptomyces sp. H1061-MY11 and Streptomyces sp.
H1089-MY 28 was resistant to tetracycline, blasticidin S, neomycin, kanamycin, streptomycin, polyoxin
and antimycin, respectively. This broad host range of
pHY12 for antibiotic-producing streptomycetes, suggests that this plasmid is useful for genetic engineering in these antibiotic-producing, and hence, resistant hosts. We measured plasmid copy-number
according to the method reported elsewhere [7].
The number of copies of pHY12 in both strains
H1061-MY11 and H1089-MY28, appeared to be
about 80.
We deleted a region non-essential for replication,
by digesting the plasmid with BamHI and BclI. The
resulting large fragment (4.3 kb) was ligated to the
BclI-cleaved pIJ702 containing the Ts-resistance gene
(Fig. 1) to generate pSY10. The plasmid number of
copies was 70^80 in S. lividans 66. The nucleotide
sequence of the 4261-bp DNA fragment except
Ts-resistance gene on pSY10, was determined and
deposited in the DNA Data Bank of Japan
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vector uses the thiostrepton (Ts)-resistance gene and
an operon responsible for synthesis of melanin pigment (mel), as selective genetic markers. The mel is
convenient due to its cloning sites which are insertionally inactivated. However, it has been reported
that Streptomyces species such as S. jumonjinensis, S.
clavuligerus and S. kasugaensis, transformed with
pIJ702, become resistant to Ts, but do not produce
melanin pigment [8]. As shown in the present study,
S. griseus transformed with pIJ702, also exhibited
the same phenotypic characteristics as the above
strains. Thus, not all Ts-resistant Streptomyces
strains, transformed with pIJ702, express the mel,
suggesting that the pIJ702 mel is not useful as an
insertional inactivated-marker gene.
Tyrosinase (o-diphenenol:oxygen oxidoreductase,
EC 1.14.18.1), a copper-containing monooxygenase,
is responsible for the production of melanin pigment
from L-tyrosine [9]. Since we found that among the
melanin pigment-producing streptomycetes S. castaneoglobisporus HUT6202 produces a greater amount
of di¡usible melanin pigment, mel, which consists of
the tyrosinase gene and an additional open reading
frame (378 bp), has been cloned from its chromosomal DNA [10]. The tyrosinase activity of S. lividans
carrying this mel was 110-fold higher than that of the
same host transformed with pIJ702 [10].
In the present study, we found that S. castaneoglobisporus HUT6202 harbors a high copy plasmid,
designated pHY6202. We constructed a cloning vector that should be useful in Streptomyces hosts in
which the pIJ702 mel is not expressed, by utilizing
the pHY6202 rep and the S. castaneoglobisporus mel.
K. Ikeda et al. / FEMS Microbiology Letters 168 (1998) 195^199
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Fig. 1. Construction strategy of pSY10CMM and its restriction enzyme map. mel, the operon encoding the synthesis of melanin pigment ;
tsr, thiostrepton resistance gene; rep, the gene encoding a protein essential for replication; spd, an essential gene for e¡ective plasmid
transfer within recipient cell ; orf68, the open reading frame, which is unidenti¢ed, consists of 68 amino acids; ori, replication origin. The
nucleotide sequence was deposited in the DDBJ databases under accession no. AB016241.
(DDBJ) databases under the accession number
AB016241. The results of this sequence analysis
and an insertional experiment of a DNA segment
into the BglII site on pSY10 suggest that the rep
and the replication origin of this plasmid are located
near BglII site (Fig. 1).
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K. Ikeda et al. / FEMS Microbiology Letters 168 (1998) 195^199
pSY10CMM has two unique restriction sites, SphI
and BamHI, in the mel.
3.2. Use of pSY10CMM as a cloning vector
Since the mel from S. castaneoglobisporus has two
SphI sites [9], we substituted one of these SphI sites
with a BamHI site generated by using the altered
sites II-in vitro mutagenesis kit (Promega, USA)
and a TGCCGGTAGGATCCGTCGTCACC primer. The mutant mel, digested with SmaI, was inserted
into pSY10 blunt ended with T4 DNA polymerase
after digestion with SphI. This new cloning vector
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Fig. 2. Melanin pigment production by S. lividans 66 (A) and S.
griseus PSR-2 (B) transformed with pSY10CMM or pIJ702.
These strains were grown on TYR-FB medium containing 50 Wg
Ts ml31 [10] at 28³C for 3 days.
Fig. 2A shows that S. lividans 66 transformed with
pSY10CMM produces a larger amount of melanin
pigment than the same strain harboring pIJ702. In
fact, the tyrosinase activity of the transformed cell
was at approximately a 110-fold higher level than
that of the same host carrying pIJ702. Fig. 2B shows
that when pIJ702 was introduced into a streptomycin-high level resistant mutant PSR-2, derived from
streptomycin-producing S. griseus [15], its transformed cells became resistant to Ts, but did not produce melanin pigment. In contrast, pSY10CMMtransformed S. griseus PSR-2 produced the melanin
pigment (Fig. 2B). These results show that
pSY10CMM can be used as cloning vector using
insertional inactivation in S. griseus, in which
pIJ702 cannot be used.
In order to ascertain whether pSY10CMM is useful as a cloning vector, a 3-kb BamHI segment including bleomycin (Bm)-resistant gene from Bm-producing S. verticillus ATCC15003 was subcloned into
the BamHI site of the pSY10CMM. S. lividans 66,
transformed with the chimeric plasmid, could not
produce di¡usible melanin pigment due to insertional inactivation of the mel. The host cell was susceptible to 50 Wg Bm ml31 , whereas the transformed cell
was resistant to over 400 Wg Bm ml31 . The transformant-harbored plasmid surely had the 3-kb insert
carrying Bm-resistant gene. When S. lividans 66 was
transformed with pSY10CMM, the number of transformants obtained per 1 Wg of plasmid DNA was
over 106 , this is almost the same e¤ciency as that
with pIJ702. The copy-number of pSY10CMM in S.
lividans 66 was estimated to be approximately 70 per
host chromosome.
We previously constructed a promoter-probe vector for E. coli using the mel from S. antibioticus as a
reporter gene [16]. The highly expressed mel from S.
castaneoglobisporus may contribute to the construction of a new promoter-probe vector which can be
used to isolate some weak promoters. The usefulness
of pSY10CMM would be enhanced by increasing the
number of its unique cloning sites.
In conclusion, pSY10CMM, is a more versatile
K. Ikeda et al. / FEMS Microbiology Letters 168 (1998) 195^199
cloning vector than the commonly used pIJ702 because of its broad host range for antibiotic-producing Streptomyces strains and its much greater production of di¡usible melanin pigment.
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