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Applied and Environmental Microbiology, June 1999, p. 2703-2709, Vol. 65, No. 6
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Development of a Self-Cloning System for Actinomadura
verrucosospora and Identification of Polyketide Synthase Genes
Essential for Production of the Angucyclic Antibiotic
Pradimicin
Tohru
Dairi,*
Yoshimitsu
Hamano,
Tamotsu
Furumai, and
Toshikazu
Oki
Biotechnology Research Center, Toyama
Prefectural University, Kurokawa 5180, Kosugi, Toyama 939-0398, Japan
Received 23 December 1998/Accepted 11 March 1999
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ABSTRACT |
A self-cloning system for Actinomadura verrucosospora,
a producer of the angucyclic antibiotic pradimicin A (PRM A), has been developed. The system is based on reproducible and reliable
protoplasting and regeneration conditions for A. verrucosospora and a novel plasmid vector that consists of a
replicon from a newly found Actinomadura plasmid
and a selectable marker cloned from the Actinomadura strain. The system has an efficiency of more than 105
CFU/microgram of DNA. Using this system, we have cloned and identified the polyketide synthase (PKS) genes essential for PRM A
biosynthesis from A. verrucosospora. Nucleotide sequence
analysis of the 3.5-kb SalI-SphI fragment
showed that ketosynthase subunits (open reading frame 1 [ORF1] and
ORF2) of the essential PKS genes have strong similarities (59 to 89%)
to those for angucyclic antibiotic biosynthesis.
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INTRODUCTION |
More than 70% of antibiotics
(including not only antibacterial agents but also bioactive microbial
compounds) have been reported to be produced by actinomycetes, which
are composed of the genus Streptomyces (68%) and the rare
actinomycetes (32%) (19). Among the rare actinomycetes, the
genus Actinomadura is reported to be the
most predominant (19). This fact shows that this genus is
one of the most important targets in screening programs for pharmacologically active compounds.
Despite these important attributes of the genus
Actinomadura, to the best of our knowledge,
with the exception of beta-lactamase of
Actinomadura sp. strain R39 (10,
16), there is no information available on the basic aspects of
gene expression in this genus due to the lack of versatile gene
manipulation systems in the genus. In the case of
Actinomadura sp. strain R39, a vector was introduced by electrotransformation into the original strain. However,
only one transformant was obtained in several assays (10),
showing that this method is not reproducible and reliable.
We are interested in the biosynthetic genes and enzymes of pradimicin A
(PRM A), which has a unique
dihydrobenzo[a]naphthacenequinone aglycone
substituted with D-alanine and two sugars and is a potent antifungal agent (21, 23). Actinomadura
hibisca and Actinomadura verrucosospora
subsp. neohibisca are representatives of PRM A producers
(21, 23). We have cloned the putative essential polyketide
synthase (PKS) genes (pms open reading frame 1 [ORF1], ORF2, and ORF3) for PRM A biosynthesis from A. hibisca by
using oligonucleotide probes based on the conserved amino acid
sequences of other PKSs (5). The essential PKS gene consists
of the 
-ketoacyl synthase units KS
and
KS
(chain-length determination factor) and an acyl
carrier protein, which is an enzyme complex which makes the
polyketide backbone by repetitive condensation of two-carbon
units (13). A homology search of the pms ORF1, ORF2, and ORF3 showed that these ORFs have a high similarity to those
of type II essential PKS genes for aromatic antibiotic
biosynthesis. Moreover, specific DNA regions
homologous to pms genes were found with genomic Southern
hybridization in all the PRM A producers examined but not in PRM A
nonproducers (5). These results suggested that the
pms ORF1, ORF2, and ORF3 would be essential PKS genes for
PRM A biosynthesis, though we had no direct evidence for this hypothesis. Therefore, we decided to develop a self-cloning
system for Actinomadura strains to confirm
this probability by complementation or a gene inactivation
technique. In this report, we describe a newly developed self-cloning
system for A. verrucosospora and the cloning and
identification of the essential PKS genes for PRM A biosynthesis from
A. verrucosospora subsp. neohibisca.
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MATERIALS AND METHODS |
Bacterial strains and plasmids.
A. verrucosospora
subsp. neohibisca E40 (9) and its derivative JN51
(a PKS gene-deficient mutant) (9) were kindly provided by
the Bristol-Myers Squibb Pharmaceutical Research Institute (Wallingford, Conn.). Actinomadura
echinospora IFO 14042 was obtained from the Institute for
Fermentation (Osaka, Japan). Actinomadura sp. strain TPA0016, Actinomadura sp. strain
TPA0019, and Actinomadura sp. strain TPA0123
were isolated from soil in the course of the secondary metabolite
screening from actinomycetes (4). Streptomyces lividans TK23 (14) was used as a host for plasmid
construction. Plasmid pIJ702 (14) and its derivative plasmid
pSE101 (a shuttle vector for Streptomyces strains and
Escherichia coli) (3), which is usually used as a
vector for the Streptomyces strain, was used for the
integration experiment. E. coli XL1-Blue MRF' (recA1
thi endA1 supE44 gyrA46 relA1 hsdR17 lac/F'
[proAB+ lacIq
lacZ
M15::Tn10{Tetr}])
(Toyobo, Osaka, Japan) and cosmid pWE15 (Toyobo) were used for
preparation of a genomic library. E. coli XL1-Blue MRF' and plasmids pBluescriptSK(+), pBluescriptKS(+), pUC18, and pUC19 were used
for the subcloning experiment and sequencing analysis.
Media.
All actinomycetes were maintained on modified
American Type Culture Collection (ATCC) no. 5 plates (6).
V15 (9) and FR-18 (9) were used with all of the
actinomycetes as the seed and production media, respectively. Mycelia
which had been stocked in 20% glycerol at 
80°C were inoculated in
4 ml of V15 medium and were grown at 30°C with shaking for 3 days.
Next, 1.5 ml of the seed culture was transferred into 30 ml of FR-18
medium in 300-ml Erlenmeyer flasks and grown at 30°C for 3 to 5 days
on a rotary shaker. All transformed actinomycetes were cultivated in
the presence of thiostrepton (10 µg/ml; Sigma). Growth conditions for
and manipulations of E. coli were as described by Maniatis et al. (17).
Analysis of PRM A.
For analysis of PRM A,
Actinomadura strains were cultivated in
liquid medium and the products were analyzed by high-pressure liquid
chromatography (HPLC). The conditions of the HPLC analysis were
described previously (9).
DNA isolation and manipulation.
Genomic DNA and plasmid
isolations from streptomycetes were done by the method of Hopwood et
al. (14). Plasmids from E. coli were prepared by
using the Qiagen Plasmid Kit (QIAGEN, Inc., Valencia, Calif.). All
restriction enzymes, T4 ligase, and calf intestinal alkaline
phosphatase were obtained from Toyobo and used according to the
manufacturer's protocols. Transformation of E. coli with
plasmid DNA by electroporation was performed under standard conditions
by using a BTX ECM 600 electroporation system (Biotechnologies and
Experimental Research, Inc., San Diego, Calif.). The procedure for
cosmid library construction has been described previously
(7).
Screening of Actinomadura
plasmids.
Cryptic plasmids pAE042, pTPA0016, pTPA0019, and
pTPA0123 were isolated from A. echinospora IFO 14042, Actinomadura sp. strain TPA0016,
Actinomadura sp. strain TPA0019, and
Actinomadura sp. strain TPA0123, respectively.
Sequence analysis.
The 3.5-kb
SalI-SphI fragment prepared from cosmid
pPRM30 was cloned into the same sites of pUC18 and pUC19. After
construction of a series of plasmids subcloned from these plasmids,
sequencing was done by the dideoxy chain termination method of Sanger
et al. (24) with an automatic DNA sequencer (model 4000L;
LI-COR).
Hybridization.
The conditions employed for colony
hybridization with the 1.7-kb BglII fragment as a probe,
which contains a putative essential PKS gene cloned from A. hibisca (5) and which was 32P labeled
(2 × 108 cpm/µg) with a nick translation kit
(Takara Syuzo, Kyoto, Japan), were as follows. A nylon membrane
(GeneScreen Plus; Dupont, Boston, Mass.) with immobilized DNA was
prehybridized in 6× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium
citrate) buffer containing 5× Denhardt's solution, 0.5% sodium
dodecyl sulfate (SDS), and 100 µg of heat-denatured salmon sperm
DNA/ml at 65°C for 4 h. For overnight hybridization, the
same buffer and temperature conditions were used. The filter was washed
twice with 0.3× SSC and 0.1% SDS buffer at 65°C for 1 h.
Preparation and transformation of protoplasts of strain E40 and
its derivatives. (i) Protoplast preparation.
Frozen stock mycelia
in 20% glycerol were inoculated into 4 ml of V15 medium and cultivated
to the stationary phase (60 h). Portions (0.5 ml) of this culture
were transferred to 30 ml of V15-P medium, consisting of sucrose (20%
[wt/vol]), glucose (3%), Bacto soyton (1.5%; Difco), glycine
(0.3%), CaCl2 · 2H2O (0.04%), and
MgCl2 · 6H2O (0.1%) (pH 7.2) and grown
to the early stationary phase. Mycelia were collected by centrifugation
(5,000 × g, 10 min), washed with modified P medium
(the composition of which was the same as that described by Hopwood et
al. [14] except for the sucrose concentration
[20%]), and then incubated in 10 ml of the same medium with lysozyme
(2.5 mg/ml) and N-acetylmuramidase (0.05 mg/ml; Seikagaku
Kogyo, Tokyo, Japan) at 32°C for 3 h. Protoplast formation was
monitored microscopically. Protoplasts were recovered by centrifugation
at 1,800 × g and 4°C for 10 min and washed twice with ice-cold modified P medium. They were gently resuspended in 5 ml
of the same medium and stored at 80°C.
(ii) Transformation.
For transformation, the following
regeneration medium (RAM-SM medium) has been developed. RAM-SM medium
was prepared by mixing the following four separately autoclaved
solutions: 983 ml of nutrient solution (200 g of sucrose, 10 g of
glucose, 2 g of glycerol, 7.5 g of yeast extract [Difco],
Casamino Acids [Difco], 1.5 g of L-asparagine,
5.2 g of MOPS [morpholinepropanesulfonic acid], 20 g of
Bacto agar [Difco] [pH 7.2]), 6 ml of 5 M CaCl2
· 2H2O, 1 ml of 0.5% KH2PO4, and
the supernatant of the sonicate of strain E40 prepared from a 50-ml
culture. The supernatant of the sonicate was prepared by sonication (in
10 ml of cold water) of the mycelia grown in 50 ml of V15 medium with a
pencil-type automatically tuned ultrasonic disruptor (model UD200;
output, 5 to 6; TOMY SEIKO Co., Ltd., Tokyo, Japan) at 30-s intervals
for a total of 3 min and then centrifugation at 10,000 × g and 4°C for 10 min. Plates were dried under laminar flow for
30 min and were immediately used. Transformation was done with plasmid
DNA in 10 µl of Tris-EDTA (TE) buffer to which a 100-µl suspension
of 109 to 1010 protoplasts was added. After the
mixture was left for 1 min at room temperature, 440 µl of 25%
(vol/vol) polyethylene glycol (PEG) 4000 with T medium was added and
mixed gently by pipetting. The mixture was diluted with modified P
medium, and 100-µl samples were plated on RAM-SM medium. These plates
were incubated at 30°C for 3 days and were then overlaid with 2.5 ml
of nutrient soft agar containing thiostrepton (final concentration, 10 µg/ml).
Nucleotide sequence accession number.
The nucleotide
sequence reported in this paper will appear in the DDBJ, EMBL, and
GenBank nucleotide sequence databases under accession no. AB019690.
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RESULTS AND DISCUSSION |
Protoplasting and regeneration of
Actinomadura strains.
Previously, we
isolated five Actinomadura strains
producing PRMs (4). We chose A. verrucosospora subsp. neohibisca E40 as the host for
DNA manipulations in this study because it does not harbor any
detectable cryptic plasmids and is the original strain of the
previously isolated PRM-nonproducing mutants (9). Moreover, because the strain was sensitive to many antibiotics, including thiostrepton, streptomycin, erythromycin, neomycin, and
kanamycin, resistance genes to these antibiotics were thought to be
proper selectable markers of the vectors for the strain. Recently, we
have developed the protocols for protoplast formation and regeneration
of the protoplast of Actinomadura strains
(4), almost all of which were extraordinarily resistant to
lysozyme. In the case of strain E40, we found that mycelia grown in the presence of sucrose (30%), the addition of which in preculture was
reported to be effective for protoplast formation of some Streptomyces strains (15), became sensitive to
lysozyme. The addition of sucrose plus glycine (0.3%) in the medium
was most effective (4). As for lytic enzymes, the use of
both lysozyme and N-acetylmuramidase (1 mg/ml) was even more
effective than the use of lysozyme alone (4). However,
we could obtain no transformants of strain E40 with ligation mixtures
that were prepared by ligating a replicon from a newly found
Actinomadura plasmid and a selectable
marker, probably because of low regeneration efficiency of the
protoplast (2.7%) on RAM-S medium (a previously described
regeneration medium) (4). Therefore, several parameters were
examined to try to improve the ratio.
With regard to the preparation of protoplasts, substances which
accelerate the protoplast formation when added to the culture medium
were investigated. Though we examined dozens of compounds, including
antibiotics inhibiting cell wall biosynthesis,
detergents, amino acids and sugar alcohols, we were not able to find
any effective substances other than sucrose as described previously
(4). However, we found that the regeneration ratio of
protoplasts, which were prepared from mycelia grown in the presence of
20% sucrose, is higher than that of protoplasts prepared from mycelia grown in the presence of 30% sucrose, though prolonged incubation (more than 3 h) with lytic enzymes was needed to complete
protoplasting. Next, we examined the condition of treatment of lytic
enzyme. When a high concentration (1 mg/ml) of
N-acetylmuramidase was used for preparation of the
protoplasts, the regeneration was found to be slightly inhibited,
though the protoplasts were washed five times with modified P
medium to remove N-acetylmuramidase. The optimal
concentration of N-acetylmuramidase was determined to be
0.05 mg/ml. The concentration of lysozyme had no effect on regeneration
of protoplasts in the range between 0.5 and 10 mg/ml. The suitable
temperature of treatment with lytic enzymes was determined to be
32°C. These modified protocols provided us with a regeneration rate
of at least 15% on RAM-S medium by several trials.
To increase the regeneration ratio of the protoplasts of strain
E40, several parameters of the regeneration medium were also
modified.
It was reported that the addition of cell extracts of
Micromonospora strains to the regeneration medium was
effective
to increase the regeneration ratio of the protoplasts of
these
strains (
12). This was also the case for
Actinomadura strains,
and the regeneration
ratio was increased by 30% when cell extracts
of strain E40 were added
to the regeneration medium. Though we
do not know what materials
in the cell extracts increased the
regeneration ratio of the
protoplasts, some high-molecular-weight
materials were thought to be
candidates because the cell extracts
after filtration with
CENTRIPREP-10 (Amicon Inc., Beverly, Mass.),
which cut off those with
molecular weights less than 10,000, were
still effective. Brief drying
of about 30 min in laminar flow
(about 3% reduction of medium weight)
was also effective in increasing
the regeneration ratio. The most
important parameter in the regeneration
medium was the type of buffer
used for pH stabilization. When
MOPS was used as a pH stabilizer
instead
of TES [
N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic
acid],
which is usually used as a pH stabilizer of the regeneration
medium of
Streptomyces strains (
14), the regeneration ratio
was increased threefold. Finally, more than 60% of the protoplasts
prepared under the optimized conditions could be regenerated on
a
modified regeneration medium (RAM-SM).
Construction of Actinomadura vectors
and transformation of strain E40.
In our preliminary experiment,
strain E40 could not be transformed by pIJ61 (14), pGM9
(20), and pIJ702 (14), the typical cloning
vectors for Streptomyces strains, with the optimized
protoplasting and regeneration conditions as described above and with
the use of PEG by the method used for S. lividans
(14). This led us to construct vectors of a new type
harboring replicons of Actinomadura strains. Cryptic plasmids were screened from our culture stocks and fresh soil isolates of
Actinomadura strains. We found four new
plasmids (pAE042, pTPA0016, pTPA0019, and pTPA0123) by analysis using
agarose gel electrophoresis. Judging from the physical maps of
these plasmids, they were thought to be different from one another.
Strain E40 was sensitive to thiostrepton, and we therefore first tried
to construct cloning vectors that had the thiostrepton
resistance gene
(
tsr) prepared from pIJ702 as a selectable marker.
To
make cloning vectors harboring
tsr, we completely digested
the newly found four plasmids with
BglII or
BamHI. The 1.0-kb
BclI fragment carrying the
tsr gene prepared from pIJ702 was ligated
with these
digested plasmids and introduced into the protoplasts
of strain E40
with the assistance of PEG by the method used for
the
S. lividans system (
14). Ten to 50 thiostrepton-resistant
transformants were obtained in a single
experiment only when
BglII
or
BamHI digests of
pTPA0123 (one of the cryptic plasmids; Fig.
1) were used as replicons. The
plasmids isolated from thiostrepton-resistant
colonies had
the structures we had expected (pTPA0123TP1 and
pTPA0123TP2;
Fig.
1). Next, we wanted a marker originating
from
Actinomadura strains to produce a
cloning system of generically homologous
backgrounds. Because
Actinomadura sp. strain TPA0019 was
moderately
resistant to thiostrepton (MIC, 50 µg/ml), the
genomic DNA of
this strain was used as a source of the
thiostrepton resistance
gene. The total genomic DNA
of strain TPA0019 was digested with
BamHI,
BclI, and
BglII; ligated with
BglII-digested pTPA0123;
and used to transform strain E40.
Thiostrepton-resistant transformants
were obtained only when the
BglII-digested genomic DNA of strain
TPA0019 was
used. All transformants examined had plasmids of the
same size
(9.7 kb). One of these plasmids was selected and named
pTPA0123TPA
(Fig.
1).
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FIG. 1.
Construction of plasmid vectors for A. verrucosospora E40 by using the cryptic plasmid of
Actinomadura sp. strain TPA0123 and the
thiostrepton resistance genes (tsr). A cryptic plasmid,
pTPA0123, digested with BglII or BamHI was
treated with calf intestinal alkaline phosphatase, ligated with a
1.0-kb BclI fragment of pIJ702 containing tsr,
and introduced into the protoplasts of A. verrucosospora
E40. Thiostrepton-resistant transformants were found to harbor
pTPA0123TP1 and pTPA0123TP2. For construction of pTPA0123TPA, the
genomic DNA of Actinomadura sp. strain
TPA0019 digested with BglII was ligated with
BglII-digested pTPA0123 and used to transform A. verrucosospora E40. All thiostrepton-resistant transformants
examined harbored 9.7-kb plasmids.
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The experiments for the construction of vectors described above
indicated that the PEG-assisted protocol for the
Streptomyces strain worked in strain E40. We optimized the
transformation conditions
with pTPA0123TP1 and pTPA0123TPA. By
tuning the type and the concentration
of PEG and optimizing the
timing of antibiotic selection, we could
obtain 10
5
transformants/µg of DNA with both pTPA0123TP1 and
pTPA0123TPA,
which were prepared from strain E40. The strains
harboring pTPA0123TP1
or pTPA0123TPA were passed through three cycles
of sporulation
and germination on modified ATCC no. 5 plates in the
presence
or absence of thiostrepton. At each cycle, spores were
collected
and checked for the presence of plasmids by plating them on
modified
ATCC no. 5 plates with or without thiostrepton. The plasmids
were
almost perfectly maintained in the presence of the antibiotic.
The
loss of the plasmid in the absence of the antibiotic is less
than 30%
even after three
cycles.
Cloning of the essential PKS genes for PRM A
biosynthesis.
Recently, we cloned the putative
essential PKS genes for PRM A biosynthesis from A. hibisca by using oligonucleotide probes based on the
conserved amino acid sequences of other PKS genes and found by
genomic Southern hybridization that there are specific DNA
regions homologous to pms genes in the genome of
strain E40 (5). Therefore, we attempted to clone the PKS
genes for PRM A biosynthesis from strain E40 by using the
DNA fragment containing the putative essential PKS genes from A. hibisca as a probe. Subsequently, we tried to obtain the
direct evidence by complementation and gene inactivation. To clone the
hybridized fragment and its flanking region simultaneously, a cosmid
library in E. coli of strain E40 DNA partially digested with
Sau3AI was screened by colony hybridization. Among them,
pPRM30 was selected for further analysis. The 7.5-kb PstI
fragment that hybridized to the probe was subcloned into pBluescriptSK(+) and used for further analysis.
Nucleotide sequence of the DNA fragment hybridized to the
probe.
To examine whether the DNA fragment hybridized to the
probes carries the PKS gene for biosynthesis of PRM A, the
nucleotide sequence of the 3.5-kb SalI-SphI
fragment containing the hybridized region (Fig.
2) was determined. Computer analysis of
the DNA sequence, using Frame Analysis (2), revealed at
least three complete ORFs (ORF1 to -3), which were all oriented in the
same direction. We searched the databases with their translated
products by means of the sequence similarity search programs BLAST
(22) and FASTA (1). As we had expected, ORF1,
ORF2, and ORF3 products show strong similarities (89, 82, and 71%
identity, respectively; Fig. 3) with
those of the putative essential PKS genes for PRM A
biosynthesis, which were previously cloned from
A. hibisca (5) and used as a probe in this study.
Moreover, ORF1 and ORF2 products were found to be significantly similar
to ketosynthase-
and - responsible for urdamycin A (73 and
59% identity, respectively) (8) and jadomycin B (69 and 59% identity, respectively) (11)
biosynthesis. On the other hand, ORF3, encoding an
acyl carrier protein, is less similar to urdamycin A and jadomycin
B biosynthetic genes (40 and 41% identity). Urdamycin A and jadomycin
B also have angucyclic structures, and the sequence similarities of
ketosynthases might therefore reflect the features of biosynthetic
genes for angular-type antibiotics.
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FIG. 2.
Construction of plasmids used for complementation and
gene inactivation experiments. The 7.5-kb PstI fragment
(open box) prepared from cosmid pPRM30 containing essential PKS genes
for PRM A biosynthesis was ligated with pTPA0123TP1, which
was digested with PstI and treated with calf intestinal
alkaline phosphatase. S. lividans TK23
(14) was transformed with the ligation mixture. A
recombinant plasmid (pPRM123) was screened among the
thiostrepton-resistant transformants and used to transform a PRM
A-nonproducing mutant, JN51. For gene inactivation experiments, pSE101
(a shuttle vector constructed with pIJ702 and pUC19) (3) was
digested with BglII and SacI, and the resulting
large fragment (6.6 kb) was ligated with the 0.7-kb
BamHI-SacI fragment (black box) carrying the
internal region of ORF1. The ligation mixture was used to transform
S. lividans TK23. A recombinant plasmid (pIPB001) was
isolated from the transformant and used to transform A. verrucosospora E40. The 3.5-kb SalI-SphI
fragment (hatched box) used for sequencing analysis is also shown.
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FIG. 3.
Alignment of the amino acid sequences of the essential
PKS gene products from A. verrucosospora E40 with those from
A. hibisca P-157-2. Deduced amino acid sequences of ORF1
(A), ORF2 (B), and ORF3 (C) of strain E40 (lines labeled E) and of
strain P-157-2 (lines labeled P) are shown. The colons and periods
indicate identical and similar residues, respectively.
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Complementation and gene inactivation of PKS genes.
To explore
whether the DNA fragment cloned in this study would contain the
essential PKS genes for PRM A biosynthesis, the 7.5-kb PstI fragment that hybridized to the probe and
contained the essential PKS genes was cloned into the same site
of pTPA0123TP1 to give pPRM123 (Fig. 2) and was introduced
into mutant JN51, which was derived from strain E40 by
N-methyl-N'-nitro-N-nitrosoguanidine treatment (9). Mutant JN51 produced no intermediates
as judged by thin-layer chromatography and HPLC analysis and always
acted as a converter during the cosynthesis study (9),
suggesting that this strain is not a mutant defective in the regulatory
gene but rather a mutant deficient in the essential PKS
genes. We constructed pPRM123 in S. lividans
and then introduced it into strain JN51, because S. lividans was found to be able to maintain pTPA0123TP1 and was
tolerant of accepting the DNA fragments prepared from E. coli. All transformants of mutant JN51 harboring the
thus-constructed pPRM123 produced PRM A, which has a red
color, on the modified ATCC no. 5 plate (Fig.
4), in contrast to those harboring a
vector (pTPA0123TP1) which produced no pigments. The transformants
harboring pPRM123 were cultivated in liquid medium, and the red pigment was confirmed to be PRM A by HPLC analysis.
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FIG. 4.
Colony colors of A. verrucosospora E40 (1),
JN51 (2), JN51 harboring pPRM123 (3), and JN51 harboring a vector
(pTPA0123TP1) (4). Panels A and B show the modified ATCC no. 5 plate
and the modified ATCC no. 5 plate containing thiostrepton (10 µg/ml),
respectively.
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We also confirmed that the essential PKS genes cloned in this study
indeed are the genes for PRM A biosynthesis by a gene
disruption experiment utilizing plasmid integration. As described
above, strain E40 could not be transformed with pIJ702 though
the
tsr gene of pIJ702 could function in strain E40, suggesting
that pIJ702 is useful for an integration vector in strain E40.
The
0.7-kb
BamHI-
SacI fragment carrying the internal
sequences
of ORF1 was subcloned into the
BglII-
SacI sites of pSE101 (a shuttle
vector for
Streptomyces strains and
E. coli)
(
3) to give pIPB001
(Fig.
2). Strain E40 was
transformed with pIPB001 prepared from
S. lividans.
About 10 thiostrepton-resistant colonies were obtained
with
1 µg of plasmid. After single colony isolation in the presence
of thiostrepton (10 µg/ml), Southern blot hybridization was
performed
to confirm the integration of pIPB001 (Fig.
5). These pIPB001-integrated
strains did
not produce any PRM A either on the plate or in liquid
cultivation,
showing that the essential PKS genes cloned in this
study indeed
are the genes for PRM A biosynthesis.
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FIG. 5.
Southern blot analysis of the genomic DNA of
ORF1-inactivated strains. (A) BglII digests of DNA from
A. verrucosospora E40 (lane 1) and from ORF1-inactivated
strains (lanes 2 to 6) were subjected to Southern blot hybridization
with the 32P-labeled 0.7-kb
BamHI-SacI fragment as a probe. Arrows indicate
the hybridized fragments. (B) The hybridized fragments shown
schematically.
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In this work, we developed a practical gene cloning system for
A. verrucosospora and cloned the essential PKS genes for PRM
A
biosynthesis. Over the last few years, many type II PKS
recombinant
gene cassettes have been constructed (
13), and a
set of predictive
design rules for the generation of
polyketides was proposed on
the basis of these studies
(
13). However, it is not fully understood
how angucyclic
antibiotics represented by PRM A and jadomycin
B are synthesized
(
18). Therefore, we are now trying to examine
whether the
PKS system for angucyclic antibiotics would follow
predictive
design rules by constructing a series of expression
cassettes
containing the essential PKS genes from a PRM A producer.
These results
will be published in the near
future.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Biotechnology
Research Center, Toyama Prefectural University, Kurokawa 5180, Kosugi, Toyama 939-0398, Japan. Phone: 81-766-56-7500, ext.
561. Fax: 81-766-56-2498. E-mail: dairi{at}pu-toyama.ac.jp.
Present address: Applied Life Science Research, Tamagawa
University, 6-1-1, Tamagawa-Gakuen, Machida, Tokyo 194-8610, Japan.
| |
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Applied and Environmental Microbiology, June 1999, p. 2703-2709, Vol. 65, No. 6
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
